Retroviral vectors comprising a functional splice donor site and a functional splice acceptor site

ABSTRACT

A retroviral vector is described. The retroviral vector comprises a functional splice donor site and a functional splice acceptor site; wherein the functional splice donor site and the functional splice acceptor site flank a first nucleotide sequence of interest (“NOI”); wherein the functional splice donor site is upstream of the functional splice acceptor site; wherein the retroviral vector is derived from a retroviral pro-vector; wherein the retroviral pro-vector comprises a first nucleotide sequence (“NS”) capable of yielding the functional splice donor site and a second NS capable of yielding the functional splice acceptor site; wherein the first NS is downstream of the second NS; such that the retroviral vector is formed as a result of reverse transcription of the retroviral pro-vector.

The present invention relates to a vector.

In particular, the present invention relates to a novel system forpackaging and expressing genetic material in a retroviral particle.

More in particular, the present invention relates to a novel systemcapable of expressing a retroviral particle that is capable ofdelivering a nucleotide sequence of interest (hereinafter abbreviated as“NOI”)—or even a plurality of NOIs—to one or more target sites.

In addition, the present invention relates to inter alia a novelretroviral vector useful in gene therapy.

BACKGROUND OF THE INVENTION

Gene therapy may include any one or more of: the addition, thereplacement, the deletion, the supplementation, the manipulation etc. ofone or more nucleotide sequences in, for example, one or more targetedsites—such as targeted cells. If the targeted sites are targeted cells,then the cells may be part of a tissue or an organ. General teachings ongene therapy may be found in Molecular Biology (Ed Robert Meyers, PubVCH, such as pages 556-558).

By way of further example, gene therapy can also provide a means bywhich any one or more of: a nucleotide sequence, such as a gene, can beapplied to replace or supplement a defective gene; a pathogenicnucleotide sequence, such as a gene, or expression product thereof canbe eliminated; a nucleotide sequence, such as a gene, or expressionproduct thereof, can be added or introduced in order, for example, tocreate a more favourable phenotype; a nucleotide sequence, such as agene, or expression product thereof can be added or introduced, forexample, for selection purposes (i.e. to select transformed cells andthe like over non-transformed cells); cells can be manipulated at themolecular level to treat, cure or prevent disease conditions—such ascancer (Schmidt-Wolf and Schmidt-Wolf, 1994, Annals of Hematology69;273-279) or other disease conditions, such as immune, cardiovascular,neurological, inflammatory or infectious disorders; antigens can bemanipulated and/or introduced to elicit an immune response, such asgenetic vaccination.

In recent years, retroviruses have been proposed for use in genetherapy. Essentially, retroviruses are RNA viruses with a life cycledifferent to that of lytic viruses. In this regard, a retrovirus is aninfectious entity that replicates through a DNA intermediate. When aretrovirus infects a cell, its genome is converted to a DNA form by areverse so transcriptase enzyme. The DNA copy serves as a template forthe production of new RNA genomes and virally encoded proteins necessaryfor the assembly of infectious viral particles.

There are many retroviruses and examples include: murine leukemia virus(MLV), human immunodeficiency virus (HIV), equine infectious anaemiavirus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus(RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus(Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murinesarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avianmyelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus(AEV).

A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

Details on the genomic structure of some retroviruses may be found inthe art. By way of example, details on HIV may be found from the NCBIGenbank (i.e. Genome Accession No. AF033819).

Essentially, all wild type retroviruses contain three major codingdomains, gag, pol, env, which code for essential virion proteins.Nevertheless, retroviruses may be broadly divided into two categories:namely, “simple” and “complex”. These categories are distinguishable bythe organisation of their genomes. Simple retroviruses usually carryonly elementary information. In contrast, complex retroviruses also codefor additional regulatory proteins derived from multiple splicedmessages.

Retroviruses may even be further divided into seven groups. Five ofthese groups represent retroviruses with oncogenic potential. Theremaining two groups are the lentiviruses and the spumaviruses. A reviewof these retroviruses is presented in “Retroviruses” (1997 Cold SpringHarbour Laboratory Press Eds: S M Coffin, S M Hughes, H E Varmus pp1-25).

All oncogenic members except the human T-cell leukemia virus-bovineleukemia virus group (HTLV-BLV) are simple retroviruses. HTLV, BLV andthe lentiviruses and spumaviruses are complex. Some of the best studiedoncogenic retroviruses are Rous sarcoma virus (RSV), mouse mammarytumour virus (MMTV) and murine leukemia virus (MLV) and the human T-cellleukemia virus (HTLV).

The lentivirus group can be split even further into “primate” and“non-primate”. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIR) and bovine immunodeficiency virus (BIV).

A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

During the process of infection, a retrovirus initially attaches to aspecific cell surface receptor. On entry into the susceptible host cell,the retroviral RNA genome is then copied to DNA by the virally encodedreverse transcriptase which is carried inside the parent virus. This DNAis transported to the host cell nucleus where it subsequently integratesinto the host genome. At this stage, it is typically referred to as theprovirus. The provirus is stable in the host chromosome during celldivision and is transcribed like other cellular proteins. The provirusencodes the proteins and packaging machinery required to make morevirus, which can leave the cell by a process sometimes called “budding”.

As already indicated, each retroviral genome comprises genes called gag,pol and env which code for virion proteins and enzymes. In the provirus,these genes are flanked at both ends by regions called long terminalrepeats (LTRs). The LTRs are responsible for proviral integration, andtranscription. They also serve as enhancer-promoter sequences. In otherwords, the LTRs can control the expression of the viral gene.Encapsidation of the retroviral RNAs occurs by virtue of a psi sequencelocated at the 5′ end of the viral genome.

The LTRs themselves are identical sequences that can be divided intothree elements, which are called U3, R and U5. U3 is derived from thesequence unique to the 3′ end of the RNA. R is derived from a sequencerepeated at both ends of the RNA and U5 is derived from the sequenceunique to the 5′ end of the RNA. The sizes of the three elements canvary considerably among different retroviruses.

For ease of understanding, simple, generic structures (not to scale) ofthe RNA and the DNA forms of the retroviral genome are presented belowin which the elementary features of the LTRs and the relativepositioning of gag, pol and env are indicated.

As shown in the diagram above, the basic molecular organisation of aninfectious retroviral RNA genome is (5′) R—U5—gag, pol, env—U3-R (3′).In a defective retroviral vector genome gag, pol and env may be absentor not functional. The R regions at both ends of the RNA are repeatedsequences. U5 and U3 represent unique sequences at the 5′ and 3′ends ofthe RNA genome respectively.

Reverse transcription of the virion RNA into double stranded DNA takesplace in the cytoplasm and involves two jumps of the reversetranscriptase from the 5′ terminus to the 3′terminus of the templatemolecule. The result of these jumps is a duplication of sequenceslocated at the 5′ and 3′ends of the virion RNA. These sequences thenoccur fused in tandem on both ends of the viral DNA, forming the longterminal repeats (LTRs) which comprise R U5 and U3 regions. Oncompletion of the reverse transcription, the viral DNA is translocatedinto the nucleus where the linear copy of the retroviral genome, calleda preintegration complex (PIC), is randomly inserted into chromosomalDNA with the aid of the virion integrase to form a stable provirus. Thenumber of possible sites of integration into the host cellular genome isvery large and very widely distributed.

The control of proviral transcription remains largely with the noncodingsequences of the viral LTR. The site of transcription initiation is atthe boundary between U3 and R in the left hand side LTR (as shown above)and the site of poly (A) addition (termination) is at the boundarybetween R and U5 in the right hand side LTR (as shown above). U3contains most of the transcriptional control elements of the provirus,which include the promoter and multiple enhancer sequences responsive tocellular and in some cases, viral transcriptional activator proteins.Some retroviruses have any one or more of the following genes such astat, rev, tax and rex that code for proteins that are involved in theregulation of gene expression.

Transcription of proviral DNA recreates the full length viral RNAgenomic and subgenomic-sized RNA molecules that are generated by RNAprocessing. Typically, all RNA products serve as templates for theproduction of viral proteins. The expression of the RNA products isachieved by a combination of RNA transcript splicing and ribosomalframshifting during translation.

RNA splicing is the process by which intervening or “intronic” RNAsequences are removed and the remaining “exonic” sequences are ligatedto provide continuous reading frames for translation. The primarytranscript of retroviral DNA is modified in several ways and closelyresembles a cellular mRNA. However, unlike most cellular mRNAs, in whichall introns are efficiently spliced, newly synthesised retroviral RNAmust be diverted into two populations. One population remains unsplicedto serve as the genomic RNA and the other population is spliced toprovide subgenomic RNA.

The full-length unspliced retroviral RNA transcripts serve twofunctions: (i) they encode the gag and pol gene products and (ii) theyare packaged into progeny virion particles as genomic RNA.Sub-genomic-sized RNA molecules provide mRNA for the remainder of theviral gene products. All spliced retroviral transcripts bear the firstexon, which spans the U5 region of the 5′ LTR. The final exon alwaysretains the U3 and R regions encoded by the 3′LTR although it varies insize. The composition of the remainder of the RNA structure depends onthe number of splicing events and the choice of alternative splicesites.

In simple retroviruses, one population of newly synthesised retroviralRNA remains unspliced to serve as the genomic RNA and as mRNA for gagand pol. The other population is spliced, fusing the 5′ portion of thegenomic RNA to the downstream genes, most commonly env. Splicing isachieved with the use of a splice donor positioned upstream of gag and asplice acceptor near the 3′terminus of pol. The intron between thesplice donor and splice acceptor that is removed by splicing containsthe gag and pol genes. This splicing event creates the mRNA for envelope(Env) protein.

Typically the splice donor is upstream of gag but in some viruses, suchas ASLV, the splice donor is positioned a few codons into the gag generesulting in a primary Env translation product that includes a fewamino-terminal amino acid residues in common with Gag. The Env proteinis synthesised on membrane-bound polyribosomes and transported by thecell's vesicular traffic to the plasma membrane, where it isincorporated into viral particles.

Complex retroviruses generate both singly and multiply splicedtranscripts that encode not only the env gene products but also the setsof regulatory and accessory proteins unique to these viruses. Compexretroviruses such as the lentiviruses, and especially HIV, providestriking examples of the complexity of alternative splicing that canoccur during retroviral infection. For example, it is now known thatHIV-1 is capable of producing over 30 different mRNAs by sub-optimalsplicing from primary genomic transcripts. This selection processappears to be regulated as mutations that disrupt competing spliceacceptors can cause shifts in the splicing patterns and can affect viralinfectivity (Purcell and Martin 1993 J Virol 67: 6365-378).

The relative proportions of full-length RNA and subgenomic-sized RNAsvary in infected cells and modulation of the levels of these transcriptsis a potential control step during retroviral gene expression. Forretroviral gene expression, both unspliced and spliced RNAs must betransported to the cytoplasm and the proper ratio of spliced andunspliced RNA must be maintained for efficient retroviral geneexpression. Different classes of retroviruses have evolved distinctsolutions to this problem. The simple retroviruses, which use onlyfull-length and singly spliced RNAs regulate the cytoplasmic ratios ofthese species either by the use of variably efficient splice sites or bythe incorporation of several cis-acting elements, that have been onlypartially defined, into their genome. The complex retroviruses, whichdirect the synthesis of both singly and multiply spliced RNA, regulatethe transport and possibly splicing of the different genomic andsubgenomic-sized RNA species through the interaction of sequences on theRNA with the protein product of one of the accessory genes, such as revin HIV-1 and rex in HTLV-1.

With regard to the structural genes gag, pol and env themselves and inslightly more detail, gag encodes the internal structural protein of thevirus. Gag is proteolytically processed into the mature proteins MA(matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes thereverse transcriptase (RT), which contains both DNA polymerase, andassociated RNase H activities and integrase (IN), which mediatesreplication of the genome. The env gene encodes the surface (SU)glycoprotein and the transmembrane (TM) protein of the virion, whichform a complex that interacts specifically with cellular receptorproteins. This interaction leads ultimately to fusion of the viralmembrane with the cell membrane.

The Env protein is a viral protein which coats the viral particle andplays an essential role in permitting viral entry into a target cell.The envelope glycoprotein complex of retroviruses includes twopolypeptides: an external, glycosylated hydrophilic polypeptide (SU) anda membrane-spanning protein (TM). Together, these form an oligomeric“knob” or “knobbed spike” on the surface of a virion. Both polypeptidesare encoded by the env gene and are synthesised in the form of apolyprotein precursor that is proteolytically cleaved during itstransport to the cell surface. Although uncleaved Env proteins are ableto bind to the receptor, the cleavage event itself is necessary toactivate the fusion potential of the protein, which is necessary forentry of the virus into the host cell. Typically, both SU and TMproteins are glycosylated at multiple sites. However, in some viruses,exemplified by MLV, TM is not glycosylated.

Although the SU and TM proteins are not always required for the assemblyof enveloped virion particles as such, they play an essential role inthe entry process. In this regard, the SU domain binds to a receptormolecule, often a specific receptor molecule, on the target cell. It isbelieved that this binding event activates the membrane fusion-inducingpotential of the TM protein after which the viral and cell membranesfuse. In some viruses, notably MLV, a cleavage event, resulting in theremoval of a short portion of the cytoplasmic tail of TM, is thought toplay a key role in uncovering the full fusion activity of the protein(Brody et al 1994 J Virol 68: 4620-4627; Rein et al 1994 J Virol 68:1773-1781). This cytoplasmic “tail”, distal to the membrane-spanningsegment of TM remains on the internal side of the viral membrane and itvaries considerably in length in different retroviruses.

Thus, the specificity of the SU/receptor interaction can define the hostrange and tissue tropism of a retrovirus. In some cases, thisspecificity may restrict the transduction potential of a recombinantretroviral vector. Here, transduction includes a process of using aviral vector to deliver a non-viral gene to a target cell. For thisreason, many gene therapy experiments have used MLV. A particular MLVthat has an envelope protein called 4070A is known as an amphotropicvirus, and this can also infect human cells because its envelope protein“docks” with a phosphate transport protein that is conserved between manand mouse. This transporter is ubiquitous and so these viruses arecapable of infecting many cell types. In some cases however, it may bebeneficial, especially from a safety point of view, to specificallytarget restricted cells. To this end, several groups have engineered amouse ecotropic retrovirus, which unlike its amphotropic relativenormally only infects mouse cells, to specifically infect particularhuman cells. Replacement of a fragment of an Env protein with anerythropoietin segement produced a recombinant retrovirus which thenbinds specifically to human cells that express the erythropoietinreceptor on their surface, such as red blood cell precursors (Maulik andPatel 1997 “Molecular Biotechnology: Therapeutic Applications andStrategies” 1997 Wiley-Liss Inc. pp 45).

In addition to gag, pol and env, the complex retroviruses also contain“accessory” genes which code for accessory or auxiliary proteins.Accessory or auxiliary proteins are defined as those proteins encoded bythe accessory genes in addition to those encoded by the usualreplicative or structural genes, gag, pol and env. These accessoryproteins are distinct from those involved in the regulation of geneexpression, like those encoded by tat, rev, tax and rex. Examples ofaccessory genes include one or more of vif, vpr, vpx, vpu and nef. Theseaccessory genes can be found in, for example, HIV (see, for examplepages 802 and 803 of “Retroviruses” Ed. Coffin et al Pub. CSHL 1997).Non-essential accessory proteins may function in specialised cell types,providing functions that are at least in part duplicative of a functionprovided by a cellular protein. Typically, the accessory genes arelocated between pot and env, just downstream from env including the U3region of the LTR or overlapping portions of the env and each other.

The complex retroviruses have evolved regulatory mechanisms that employvirally encoded transcriptional activators as well as cellulartranscriptional factors. These trans-acting viral proteins serve asactivators of RNA transcription directed by the LTRs. Thetranscriptional trans-activators of the lentiviruses are encoded by theviral rat genes. Tat binds to a stable, stem-loop, RNA secondarystructure, referred to as TAR, one function of which is to apparentlyoptimally position Tat to trans-activate transcription.

As mentioned earlier, retroviruses have been proposed as a deliverysystem (otherwise expressed as a delivery vehicle or delivery vector)for inter alia the transfer of a NOI, or a plurality of NOIs, to one ormore sites of interest. The transfer can occur in vitro, ex vivo, invivo, or combinations thereof. When used in this fashion, theretroviruses are typically called retroviral vectors or recombinantretroviral vectors. Retroviral vectors have even been exploited to studyvarious aspects of the retrovirus life cycle, including receptor usage,reverse transcription and RNA packaging (reviewed by Miller, 1992 CurrTop Microbiol Immunol 158:1-24).

In a typical recombinant retroviral vector for use in gene therapy, atleast part of one or more of the gag, pol and env protein coding regionsmay be removed from the virus. This makes the retroviral vectorreplication-defective. The removed portions may even be replaced by aNOI in order to generate a virus capable of integrating its genome intoa host genome but wherein the modified viral genome is unable topropagate itself due to a lack of structural proteins. When integratedin the host genome, expression of the NOI occurs—resulting in, forexample, a therapeutic and/or a diagnostic effect. Thus, the transfer ofa NOI into a site of interest is typically achieved by: integrating theNOI into the recombinant viral vector; packaging the modified viralvector into a virion coat; and allowing transduction of a site ofinterest—such as a targeted cell or a targeted cell population.

It is possible to propagate and isolate quantities of retroviral vectors(e.g. to prepare suitable titres of the retroviral vector) forsubsequent transduction of, for example, a site of interest by using acombination of a packaging or helper cell line and a recombinant vector.

In some instances, propagation and isolation may entail isolation of theretroviral gag, pol and env genes and their separate introduction into ahost cell to produce a “packaging cell line”. The packaging cell lineproduces the proteins required for packaging retroviral DNA but itcannot bring about encapsidation due to the lack of a psi region.However, when a recombinant vector carrying a NOI and a psi region isintroduced into the packaging cell line, the helper proteins can packagethe psi-positive recombinant vector to produce the recombinant virusstock. This can be used to transduce cells to introduce the NOI into thegenome of the cells. The recombinant virus whose genome lacks all genesrequired to make viral proteins can tranduce only once and cannotpropagate. These viral vectors which are only capable of a single roundof transduction of target cells are known as replication defectivevectors. Hence, the NOI is introduced into the host/target cell genomewithout the generation of potentially harmful retrovirus. A summary ofthe available packaging lines is presented in “Retroviruses” (1997 ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 449).

The design of retroviral packaging cell lines has evolved to address theproblem of inter alia the spontaneous production of helper virus thatwas frequently encountered with early designs. As recombination isgreatly facilitated by homology, reducing or eliminating homologybetween the genomes of the vector and the helper has reduced the problemof helper virus production. More recently, packaging cells have beendeveloped in which the gag, pol and env viral coding regions are carriedon separate expression plasmids that are independently transfected intoa packaging cell line so that three recombinant events are required forwild type viral production. This reduces the potential for production ofa replication-competent virus. This strategy is sometimes referred to asthe three plasmid transfection method (Soneoka et al 1995 Nucl. AcidsRes. 23: 628-33).

Transient transfection can also be used to measure vector productionwhen vectors are being developed. In this regard, transient transfectionavoids the longer time required to generate stable vector-producing celllines and is used if the vector or retroviral packaging components aretoxic to cells. Components typically used to generate retroviral vectorsinclude a plasmid encoding the Gag/Pol proteins, a plasmid encoding theEnv protein and a plasmid containing a NOI. Vector production involvestransient transfection of one or more of these components into cellscontaining the other required components. If the vector encodes toxicgenes or genes that interfere with the replication of the host cell,such as inhibitors of the cell cycle or genes that induce apotosis, itmay be difficult to generate stable vector-producing cell lines, buttransient transfection can be used to produce the vector before thecells die. Also, cell lines have been developed using transientinfection that produce vector titre levels that are comparable to thelevels obtained from stable vector-producing cell lines (Pear et al1993, Proc Natl Acad Sci 90:8392-8396).

In view of the toxicity of some HIV proteins—which can make it difficultto generate stable HIV-based packaging cells—HIV vectors are usuallymade by transient transfection of vector and helper virus. Some workershave even replaced the HIV Env protein with that of vesicular stomatisvirus (VSV). Insertion of the Env protein of VSV facilitates vectorconcentration as HIV/VSV-G vectors with titres of 5×10⁵ (10⁸ afterconcentration) have been generated by transient transfection (Naldini etal 1996 Science 272: 263-267). Thus, transient transfection of HIVvectors may provide a useful strategy for the generation of high titrevectors (Yee et al 1994 PNAS. 91: 9564-9568).

With regard to vector titre, the practical uses of retroviral vectorshave been limited largely by the titres of transducing particles whichcan be attained in vitro culture (typically not more than 10⁸particles/ml) and the sensitivity of many enveloped viruses totraditional biochemical and physicochemical techniques for concentratingand purifying viruses.

By way of example, several methods for concentration of retroviralvectors have been developed, including the use of centrifugation (Feketeand Cepko 1993 Mol Cell Biol 13: 2604-2613), hollow fibre filtration(Paul et al 1993 Hum Gene Ther 4: 609-615) and tangential flowfiltration (Kotani et al 1994 Hum Gene Ther 5: 19-28). Although a20-fold increase in viral titre can be achieved, the relative fragilityof retroviral Env protein limits the ability to concentrate retroviralvectors and concentrating the virus usually results in a poor recoveryof infectious virions. While this problem can be overcome bysubstitution of the retroviral Env protein with the more stable VSV-Gprotein, as described above, which allows for more effective vectorconcentration with better yields, it suffers from the drawback that theVSV-G protein is quite toxic to cells.

Although helper-virus free vector titres of 10⁷ cfu/ml are obtainablewith currently available vectors, experiments can often be done withmuch lower-titre vector stocks. However, for practical reasons,high-titre virus is desirable, especially when a large number of cellsmust be infected. In addition, high titres are a requirement fortransduction of a large percentage of certain cell types. For example,the frequency of human hematopoietic progenitor cell infection isstrongly dependent on vector titre, and useful frequencies of infectionoccur only with very high-titre stocks (Hock and Miller 1986 Nature 320:275-277; Hogge and Humphries 1987 Blood 69: 611-617). In these cases, itis not sufficient simply to expose the cells to a larger volume of virusto compensate for a low virus titre. On the contrary, in some cases, theconcentration of infectious vector virions may be critical to promoteefficient transduction.

Workers are trying to create high titre vectors for use in genedelivery. By way of example, a comparison of different vector designshas proved useful in helping to define the essential elements requiredfor high-titre viral production. Early work on different retroviralvector design showed that almost all of the internal protein-encodingregions of MLVs could be deleted without abolishing the infectivity ofthe vector (Miller et al 1983 Proc Natl Acad Sci 80: 4709-4713). Theseearly vectors retained only a small portion of the 3′ end of theenv-coding region. Subsequent work has shown that all of theenv-gene-coding sequences can be removed without further reduction invector titre (Miller and Rosman 1989 Biotechnique 7: 980-990;Morgenstern and Land 1990 Nucleic Acids Res 18: 3587-3596). Only theviral LTRs and short regions adjoining the LTRs, including the segmentsneeded for plus- and minus-strand DNA priming and a region required forselective packaging of viral RNA into virions (the psi site; Mann et al1983 Cell 33: 153-159) were deemed necessary for vector transmission.Nevertheless, viral titres obtained with these early vectors were stillabout tenfold lower than the parental helper virus titre.

Additional experiments indicated that retention of sequences at the 5′end of the gag gene significantly raised viral vector titres and thatthis was due to an increase in the packaging efficiency of viral RNAinto virions (Armentano et al 1987 J Virol 61: 1647-1650; Bender et al1987 J Virol 61: 1639-1646; Adam and Miller 1988 J Virol 62: 3802-3806).This effect was not due to viral protein synthesis from the gag regionof the vector because disruption of the gag reading frame or mutatingthe gag codon to a top codon had no effect on vector titre (Bender et at1987 ibid). These experiments demonstrated that the sequences requiredfor efficient packaging of genomic RNA in MLV were larger than the psisignal previously defined by deletion analysis (Mann et al 1983 ibid).In order to obtain high titres (10⁶ to >10⁷), it was shown to beimportant that this larger, signal, called psi plus, be included inretroviral vectors. It has now been demonstrated that this signal spansfrom upstream of the splice donor to downstream of the gag start codon(Bender et al 1987 ibid). Because of this position, in spliced envexpressing transcripts this signal is deleted. This ensures that onlyfull length transcripts containing all three essential genes for virallife cycle are packaged.

In addition to manipulating the retroviral vector with a view toincreasing vector titre, retroviral vectors have also been designed toinduce the production of a specific NOI (usually a marker protein) intransduced cells. As already mentioned, the most common retroviralvector design involves the replacement of retroviral sequences with oneor more NOIs to create replication-defective vectors. The simplestapproach has been to use the promoter in the retroviral 5′ LTR tocontrol the expresssion of a cDNA encoding an NOI or to alter theenhancer/promoter of the LTR to provide tissue-specific expression orinducibility. Alternatively, a single coding region has been expressedby using an internal promoter which permits more flexibility in promoterselection.

These strategies for expression of a gene of interest have been mosteasily implemented when the NOI is a selectable marker, as in the caseof hypoxanthine-guanine phosphoribosyl transferase (hprt) (Miller et al1983 Proc Natl Acad Sci 80: 4709-4713) which facilitates the selectionof vector transduced cells. If the vector contains an NOI that is not aselectable marker, the vector can be introduced into packaging cells byco-transfection with a selectable marker present on a separate plasmid.This strategy has an appealing advantage for gene therapy in that asingle protein is expressed in the ultimate target cells and possibletoxicity or antigenicity of a selectable marker is avoided. However,when the inserted gene is not selectable, this approach has thedisadvantage that it is more difficult to generate cells that produce ahigh titre vector stock. In addition it is usually more difficult todetermine the titre of the vector.

The current methodologies used to design retroviral vectors that expresstwo or more proteins have relied on three general strategies. Theseinclude: (i) the expression of different proteins from alternativelyspliced mRNAs transcribed from one promoter; (ii) the use of thepromoter in the 5′ LTR and internal promoters to drive transcription ofdifferent cDNAs and (iii) the use of internal ribosomal entry site(IRES) elements to allow translation of multiple coding regions fromeither a single mRNA or from fusion proteins that can then be expressedfrom an open reading frame.

Vectors containing internal promoters have been widely used to expressmultiple genes. An internal promoter makes it possible to exploit thepromoter/enhancer combinations other than the viral LTR for driving geneexpression. Multiple internal promoters can be included in a retroviralvector and it has proved possible to express at least three differentcDNAs each from its own promoter (Overell et al 1988 Mol Cell Biol 8:1803-1808).

While there now exist many such modified retroviral vectors which may beused for the expression of NOIs in a variety of mammalian cells, most ofthese retroviral vectors are derived from simple retroviruses such asmurine oncoretroviruses that are incapable of transducing non-dividingcells.

By way of example, a widely used vector that employs alternativesplicing to express genes from the viral LTR SV(X) (Cepko et al 1984Cell 37: 1053-1062) contains the neomycin phosphotransferase gene as aselectable marker. The model for this type of vector is the parentalvirus, MO-MLV, in which the Gag and Gag-Pol proteins are translated fromthe full-length viral mRNA and the Env protein is made from the splicedmRNA. One of the proteins encoded by the vector is translated from thefull-length RNA whereas splicing that links the splice donor near the 5′LTR to a splice acceptor just upstream of the second gene produces anRNA from which the second gene product can be translated. One drawbackof this strategy is that foreign sequences are inserted into the intronof the spliced gene. This can affect the ratio of spliced to unsplicedRNAs or provide alternative splice acceptors that interfere withproduction of the spliced RNA encoding the second gene product (Kormanet al 1987 Proc Natl Acad Sci 84: 2150-2154). Because these effects areunpredictable, they can affect the production of the encoded genes.

Other modified retroviral vectors can be divided into two classes withregards to splicing capabilities.

The first class of modified retroviral vector, typified by the pBABEvectors (Morgenstern et al 1990 Nucleic Acid Research 18: 3587-3596),contain mutations within the splice donor (GT to GC) that inhibitsplicing of viral transcripts. Such splicing inhibition is beneficialfor two reasons: Firstly, it ensures all viral transcripts contain apackaging signal and thus all can be packaged in the producer cell.Secondly, it prevents potential aberrant splicing between viral splicedonors and possible cryptic splice acceptors of inserted genes.

The second class of modified retroviral vector, typified by both N2(Miller et at 1989 Biotechniques 7: 980-990) and the more recent MFG(Dranoff et al 1993 Proc Natl Acad Sci 19: 3979-3986), containfunctional introns. Both of these vectors use the normal splice donorfound within the packaging signal. However, their respective spliceacceptors (SAs) differ. For N2, the SA is found within the “extended”packaging signal (Bender et al 1987 ibid). For MFG, the natural SA(found within pol, see FIG. 1 thereof) is used. For both these vectors,it has been demonstrated that splicing greatly enhances gene expressionin transduced cells (Miller et al 1989 ibid; Krall et al 1996 GeneTherapy 3: 37-48). Such observations support previous findings that, ingeneral, splicing can enhance mRNA translation (Lee et al 1981 Nature294: 228-232; Lewis et al 1986 Mol Cell Biol 6: 1998-2010; Chapman et al1991 Nucleic Acids Res 19: 3979-3986). One likely reason for this isthat the same machinery involved in transcript splicing may also aid intranscript export from the nucleus.

Unlike the modified retroviral vectors described above, there has beenvery little work on alternative splicing in the retroviral lentiviralsystems which are capable of infecting non-dividing cells (Naldini et al1996 Science 272: 263-267). To date the only published lentiviralvectors are those derived from HIV-1 (Kim et al 1997 J Virol 72:811-816) and FIV (Poeschla et al 1998 Nat Med 4: 354-357). These vectorsstill contain virally derived splice donor and acceptor sequences(Naldini et al 1996 ibid).

Some alternative approaches to developing high titre vectors for genedelivery have included the use of: (i) defective viral vectors such asadenoviruses, adeno-associated virus (AAV), herpes viruses, and poxviruses and (ii) modified retroviral vector designs.

The adenovirus is a double-stranded, linear DNA virus that does not gothrough an RNA intermediate. There are over 50 different human serotypesof adenovirus divided into 6 subgroups based on the genetic sequencehomology. The natural target of adenovirus is the respiratory andgastrointestinal epithelia, generally giving rise to only mild symptoms.Serotypes 2 and 5 (with 95% sequence homology) are most commonly used inadenoviral vector systems and are normally associated with upperrespiratory tract infections in the young.

Adenoviruses are nonenveloped, regular icosohedrons. A typicaladenovirus comprises a 140 nm encapsidated DNA virus. The icosahedralsymmetry of the virus is composed of 152 capsomeres: 240 hexons and 12pentons. The core of the particle contains the 36 kb linear duplex DNAwhich is covalently associated at the 5′ ends with the Terminal Protein(TP) which acts as a primer for DNA replication. The DNA has invertedterminal repeats (ITR) and the length of these varies with the serotype.

Entry of adenovirus into cells involves a series of distinct events.Attachment of the virus to the cell occurs via an interaction betweenthe viral fibre (37 nm) and the fibre receptors on the cell. Thisreceptor has recently been identified for Ad2/5 serotypes and designatedas CAR (Coxsackie and Adeno Receptor, Tomko et al (1997 Proc Natl AcadSci 94: 3352-2258). Internalisation of the virus into the endosome viathe cellular αvβ3 and αvβ5 integrins is mediated by and viral RGDsequence in the penton-base capsid protein (Wickham et al., 1993 Cell73: 309-319). Following Internalisation, the endosome is disrupted by aprocess known as endosomolysis, an event which is believed to bepreferentially promoted by the cellular αvβ5 integrin (Wickham et al.,1994 J Cell Biol 127: 257-264). In addition, there is recent evidencethat the Ad5 fibre knob binds with high affinity to the MHC class 1 α2domain at the surface of certain cell types including human epithelialand B lymphoblast cells (Hong et al., 1997 EMBO 16; 2294-2306).

Subsequently the virus is translocated to the nucleus where activationof the early regions occurs and is shortly followed by DNA replicationand activation of the late regions. Transcription, replication andpackaging of the adenoviral DNA requires both host and viral functionalprotein machinery.

Viral gene expression can be divided into early (E) and late (L) phases.The late phase is defined by the onset of viral DNA replication.Adenovirus structural proteins are generally synthesised during the latephase. Following adenovirus infection, host cellular mRNA and proteinsynthesis is inhibited in cells infected with most serotypes. Theadenovirus lytic cycle with adenovirus 2 and adenovirus 5 is veryefficient and results in approximately 10,000 virions per infected cellalong with the synthesis of excess viral protein and DNA that is notincorporated into the virion. Early adenovirus transcription is acomplicated sequence of interrelated biochemical events but it entailsessentially the synthesis of viral RNAs prior to the onset of DNAreplication.

The Schematic diagram below is of the adenovirus genome showing therelative direction and position of early and late gene transcription:

The organisation of the adenovirus genome is similiar in all of theadenovirus groups and specific functions are generally positioned atidentical locations for each serotype studied. Early cytoplasmicmessenger RNAs are complementary to four defined, noncontiguous regionson the viral DNA. These regions are designated E1-E4. The earlytranscripts have been classified into an array of intermediate early(E1a), delayed early (E1b, E2a, E2b, E3 and E4), and intermediateregions.

The early genes are expressed about 6-8 hours after infection and aredriven from 7 promoters in gene blocks E1-4.

The E1a region is involved in transcriptional transactivation of viraland cellular genes as well as transcriptional repression of othersequences. The E1a gene exerts an important control function on all ofthe other early adenovirus messenger RNAs. In normal tisssues, in orderto transcribe regions E1b, E2a, E2b, E3 or E4 efficiently, active E1aproduct is required. However, the E1a function may be bypassed. Cellsmay be manipulated to provide E1a-like functions or may naturallycontain such functions. The virus may also be manipulated to bypass theE1a function. The viral packaging signal overlaps with the E1a enhancer(194-358 nt).

The E1b region influences viral and cellular metabolism and host proteinshut-off. It also includes the gene encoding the pIX protein (3525-4088nt) which is required for packaging of the full length viral DNA and isimportant for the thermostability of the virus. The E1b region isrequired for the normal progression of viral events late in infection.The E1b product acts in the host nucleus. Mutants generated within theE1b sequences exhibit diminished late viral mRNA accumulation as well asimpairment in the inhibition of host cellular transport normallyobserved late in adenovirus infection. E1b is required for alteringfunctions of the host cell such that processing and transport areshifted in favour of viral late gene products. These products thenresult in viral packaging and release of virions. E1b produces a 19 kDprotein that prevents apoptosis. E1b also produces a 55 kD protein thatbinds to p53. For a review on adenoviruses and their replication, see WO96/17053.

The E2 region is essential as it encodes the 72 kDa DNA binding protein,DNA polymerase and the 80 kDa precurser of the 55 kDa Terminal Protein(TP) needed for protein priming to initiate DNA synthesis.

A 19 kDa protein (gp19K) is encoded within the E3 region and has beenimplicated in modulating the host immune response to the virus.Expression of this protein is upregulated in response to TNF alphaduring the first phase of the infection and this then binds and preventsmigration of the MHC class I antigens to the epithelial surface, therebydampening the recognition of the adenoviral infected cells by thecytotoxic T lymphocytes. The E3 region is dispensible in in vitrostudies and can be removed by deletion of a 1.9 kb XbaI fragment.

The E4 region is concerned with decreasing the host protein synthesisand increasing the DNA replication of the virus.

There are 5 families of late genes and all are initiated from the majorlate promoter. The expression of the late genes includes a very complexpost-transcriptional control mechanism involving RNA splicing. The fibreprotein is encoded within the L5 region. The adenoviral genome isflanked by the inverted terminal repeat which in Ad5 is 103 bp and isessential for DNA replication. 30-40 hours post infection viralproduction is complete.

Adenoviruses may be converted for use as vectors for gene transfer bydeleting the E1 gene, which is important for the induction of the E2, E3and E4 promoters. The E1-replication defective virus may be propagatedin a cell line that provides the E1 polypeptides in trans, such as thehuman embryonic kidney cell line 293. A therapeutic gene or genes can beinserted by recombination in place of the E1 gene, Expression of thegene is driven from either the E 1 promoter or a heterologous promoter.

Even more attenuated adenoviral vectors have been developed by deletingsome or all of the E4 open reading frames (ORFs). However, certainsecond generation vectors appear not to give longer-tern geneexpression, even though the DNA seems to be maintained. Thus, it appearsthat the function of one or more of the E4 ORFs may be to enhance geneexpression from at least certain viral promoters carried by the virus.

An alternative approach to making a more defective virus has been to“gut” the virus completely maintaining only the terminal repeatsrequired for viral replication. The “gutted” or “gutless” viruses can begrown to high titres with a first generation helper virus in the 293cell line but it has been difficult to separate the “gutted” vector fromthe helper virus.

Replication-competent adenoviruses can also be used for gene therapy.For example, the E1A gene can be inserted into a first generation virusunder the regulation of a tumour-specific promoter. In thoery, followinginjection of the virus into a tumour, it could replicated specificallyin the tumour but not in the surrounding normal cells. This type ofvector could be used either to kill tumour cells directly by lysis or todeliver a “suicide gene” such as the herpes-simplex-virusthymidine-kinase gene (HSV tk) which can kill infected and bystandercells following treatment with ganciclovir. Alternatively, an adenovirusdefective only for E1b has been used specifically for antitumourtreatment in phase-1 clinical trials. The polypeptides encoded by E1bare able to block p53-mediated apoptosis, preventing the cell fromkilling itself in response to viral infection. Thus, in normal nontumourcells, in the absence of E1b, the virus is unable to block apoptosis andis thus unable to produce infectious virus and spread. In tumour cellsdeficient in p53, the E1b defective virus can grow and spread toadjacent p53-defective tumour cells but not to normal cells. Again, thistype of vector could also be used to deliver a therapeutic gene such asHSV tk.

The adenovirus provides advantages as a vector for gene delivery overother gene therapy vector systems for the following reasons:

It is a double stranded DNA nonenveloped virus that is capable of invivo and in vitro transduction of a broad range of cell types of humanand non-human origin. These cells include respiratory airway epithelialcells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes,primary mammary epithelial cess and post-mitotically terminallydifferentiated cells such as neurons (with perhaps the importantexception of some lymphoid cells including monocytes).

Adenoviral vectors are also capable of transducing non dividing cells.This is very important for diseases, such as cystic fibrosis, in whichthe affected cells in the lung epithelium, have a slow turnover rate. Infact, several trials are underway utilising adenovirus-mediated transferof cystic fibrosis transporter (CFTR) into the lungs of afflicted adultcystic fibrosis patients.

Adenoviruses have been used as vectors for gene therapy and forexpression of heterologous genes. The large (36 kilobase) genome canaccommodate up to 8 kb of foreign insert DNA and is able to replicateefficiently in complementing cell lines to produce very high titres ofup to 10¹². Adenovirus is thus one of the best systems to study theexpression of genes in primary non-replicative cells.

The expression of viral or foreign genes from the adenovirus genome doesnot require a replicating cell. Adenoviral vectors enter cells byreceptor mediated endocytosis. Once inside the cell, adenovirus vectorsrarely integrate into the host chromosome. Instead, it functionsepisomally (independently from the host genome) as a linear genome inthe host nucleus. Hence the use of recombinant adenovirus alleviates theproblems associated with random integration into the host genome.

There is no association of human malignancy with adenovirus infection.Attenuated adenoviral strains have been developed and have been used inhumans as live vaccines.

However, current adenoviral vectors suffer from some major limitationsfor in vivo therapeutic use. These include: (i) transient geneexpression- the adenoviral vector generally remains episomal and doesnot replicate so that it is not passed onto subsequent progeny (ii)because of its inability to replicate, target cell proliferation canlead to dilution of the vector (iii) an immunological response raisedagainst the adenoviral proteins so that cells expressing adenoviralproteins, even at a low level, are destroyed and (iv) an inability toachieve an effective therapeutic index since in vivo delivery leads toan uptake of the vector and expression of the delivered genes in only aproportion of target cells.

If the features of adenoviruses can be combined with the geneticstability of retro/lentiviruses then essentially the adenovirus can beused to transduce target cells to become transient retroviral producercells that can stably infect neighbouring cells.

SUMMARY OF THE INVENTION

The present invention seeks to provide a novel retroviral vector.

In particular, the present invention seeks to provide a novel retroviralvector capable of providing efficient expression of a NOI—or even aplurality of NOIs—at one or more desired target sites.

The present invention also seeks to provide a novel system for preparinghigh titres of vector virion which incorporates safety features for invivo use and which is capable of providing efficient expression of aNOI—or even a plurality of NOIs—at one or more desired target sites.

According to a first aspect of the present invention, there is provideda retroviral vector comprising a functional splice donor site and afunctional splice acceptor site; wherein the functional splice donorsite and the functional splice acceptor site flank a first nucleotidesequence of interest (“NOI”); wherein the functional splice donor siteis upstream of the functional splice acceptor site; wherein theretroviral vector is derived from a retroviral pro-vector; wherein theretroviral pro-vector comprises a first nucleotide sequence (NS) capableof yielding the functional splice donor site and a second NS capable ofyielding the functional splice acceptor site; wherein the first NS isdownstream of the second NS; such that the retroviral vector is formedas a result of reverse transcription of the retroviral pro-vector.

According to a second aspect of the present invention, there is provideda retroviral vector wherein the retroviral pro-vector comprises aretroviral packaging signal; and wherein the second NS is locateddownstream of the retroviral packaging signal such that splicing ispreventable at a primary target site.

According to a third aspect of the present invention, there is provideda retroviral vector wherein the second NS is placed downstream of thefirst NOI such that the first NOI is capable of being expressed at aprimary target site.

According to a fourth aspect of the present invention, there is provideda retroviral vector wherein the second NS is placed upstream of amultiple cloning site such that one or more additional NOIs may beinserted.

According to a fifth aspect of the present invention, there is provideda retroviral vector wherein the second NS is a nucleotide sequencecoding for an immunological molecule or a part thereof.

According to a sixth aspect of the present invention, there is provideda retroviral vector wherein the immunological molecule is animmunoglobulin.

According to a seventh aspect of the present invention, there isprovided a retroviral vector wherein the second NS is a nucleotidesequence coding for an immunoglobulin heavy chain variable region.

According to a eight aspect of the present invention, there is provideda retroviral vector wherein the vector additionally comprises afunctional intron.

According to a ninth aspect of the present invention, there is provideda retroviral vector wherein the functional intron is positioned so thatit is capable of restricting expression of at least one of the NOIs in adesired target site.

According to a tenth aspect of the present invention, there is provideda retroviral vector wherein the target site is a cell.

According to a eleventh aspect of the present invention, there isprovided a retroviral vector wherein the vector or pro-vector isderivable from a murine oncoretrovirus or a lentivirus.

According to a twelfth aspect of the present invention, there isprovided a retroviral vector wherein the vector is derivable from MMLV,MSV, MMTV, HIV-1 or EIAV.

According to a thirteenth aspect of the present invention, there isprovided a retroviral vector wherein the retroviral vector is anintegrated provirus.

According to a fourteenth aspect of the present invention, there isprovided a retroviral particle obtainable from a retroviral vector.

According to a fifteenth aspect of the present invention, there isprovided a cell transfected or transduced with a retroviral vector.

According to a sixteenth aspect of the present invention there isprovided a retroviral vector or a viral particle or a cell for use inmedicine.

According to a seventeenth aspect of the present invention there isprovided a retroviral vector or a viral particle or a cell for themanufacture of a pharmaceutical composition to deliver one or more NOIsto a target site in need of same.

According to a eighteenth aspect of the present invention there isprovided a method comprising transfecting or transducing a cell with aretroviral vector or a viral particle or by use of a cell.

According to a nineteenth aspect of the present invention there isprovided a delivery system for a retroviral vector or a viral particleor a cell wherein the delivery system comprises one or morenon-retroviral expression vector(s), adenoviruse(s), or plasmid(s) orcombinations thereof for delivery of an NOI or a plurality of NOIs to afirst target cell and a retroviral vector for delivery of an NOI or aplurality of NOIs to a second target cell.

According to a twentieth aspect of the present invention there isprovided a retroviral pro-vector.

According to a twenty first aspect of the present invention there isprovided the use of a functional intron to restrict expression of one ormore NOIs within a desired target cell.

According to a twenty second aspect of the present invention there isprovided the use of a reverse transcriptase to deliver a first NS fromthe 3′ end of a retroviral pro-vector to the 5′ end of a retroviralvector.

According to a twenty third aspect of the present invention there isprovided a hybrid viral vector system for in vivo gene delivery, whichsystem comprises one or more primary viral vectors which encode asecondary viral vector, the primary vector or vectors capable ofinfecting a first target cell and of expressing therein the secondaryviral vector, which secondary vector is capable of transducing asecondary target cell.

According to a twenty fourth aspect of the present invention there isprovided a hybrid viral vector system wherein the primary vector isobtainable from or is based on a adenoviral vector and/or the secondaryviral vector is obtainable from or is based on a retroviral vectorpreferably a lentiviral vector.

According to a twenty fifth aspect of the present invention there isprovided a hybrid viral vector system wherein the lentiviral vectorcomprises or is capable of delivering a split-intron configuration.

According to a twenty sixth aspect of the present invention there isprovided a lentiviral vector system wherein the lentiviral vectorcomprises or is capable of delivering a split-intron configuration.

According to a twenty seventh aspect of the present invention there isprovided an adenoviral vector system wherein the adenoviral vectorcomprises or is capable of delivering a split-intron configuration.

According to a twenty eighth aspect of the present invention there isprovided vectors or plasmids basd on or obtained from any one or more ofthe entities presented as pE1sp1A, pCI-Neo, pE1RevE, pE1HORSE3.1,pE1PEGASUS4, pCI-Rab, pE1Rab.

According to a twenty ninth aspect of the present invention there isprovided a retroviral vector capable of differential expression of NOIsin target cells.

Another aspect of the present invention includes a hybrid viral vectorsystem for in vivo gene delivery, which system comprises a primary viralvector which encodes a secondary viral vector, the primary vectorcapable of infecting a first target cell and of expressing therein thesecondary viral vector, which secondary vector is capable of transducinga secondary target cell, wherein the primary vector is obtainable fromor is based on a adenoviral vector and the secondary viral vector isobtainable from or is based on a retroviral vector preferably alentiviral vector.

Another aspect of the present invention includes a hybrid viral vectorsystem for in vivo gene delivery, which system comprises a primary viralvector which encodes a secondary viral vector, the primary vectorcapable of infecting a first target cell and of expressing therein thesecondary viral vector, which secondary vector is capable of transducinga secondary target cell, wherein the primary vector is obtainable fromor is based on a adenoviral vector and the secondary viral vector isobtainable from or is based on a retroviral vector preferably alentiviral vector; wherein the viral vector system comprises afunctional splice donor site and a functional splice acceptor site;wherein the functional splice donor site and the functional spliceacceptor site flank a first nucleotide sequence of interest (“NOI”);wherein the functional splice donor site is upstream of the functionalsplice acceptor site; wherein the retroviral vector is derived from aretroviral pro-vector; wherein the retroviral pro-vector comprises afirst nucleotide sequence (“NS”) capable of yielding the functionalsplice donor site and a second NS capable of yielding the functionalsplice acceptor site; wherein the first NS is downstream of the secondNS; such that the retroviral vector is formed as a result of reversetranscription of the retroviral pro-vector.

Preferably the retroviral pro-vector comprises a third NS that isupstream of the second nucleotide sequence; wherein the third NS iscapable of yielding a non-functional splice donor site.

Preferably the retroviral vector further comprises a second NOI; whereinthe second NOI is downstream of the functional splice acceptor site.

Preferably the retroviral pro-vector comprises the second NOI; whereinthe second NOI is downstream of the second nucleotide sequence.

Preferably the second NOI, or the expression product thereof, is orcomprises a therapeutic agent or a diagnostic agent.

Preferably the first NOI, or the expression product thereof, is orcomprises any one or more of an agent conferring selectablity (e.g. amarker element), a viral essential element, or a part thereof, orcombinations thereof.

Preferably the first NS is at or near to the 3′ end of a retroviralpro-vector; preferably wherein the 3′ end comprises a U3 region and an Rregion; and preferably wherein the first NS is located between the U3region and the R region.

Preferably the U3 region and/or the first NS of the retroviralpro-vector comprises an NS that is a third NOI; wherein the NOI is anyone or more of a transcriptional control element, a coding sequence or apart thereof.

Preferably the first NS is obtainable from a virus.

Preferably the first NS is an intron or a part thereof.

Preferably the intron is obtainable from the small t-intron of SV40virus.

Preferably the vector components are regulated. In one preferred aspectof the invention, the vector components are regulated by hypoxia.

In another preferred aspect of the invention, the vector components areregulated by tetracycline on/off system.

Thus, the present invention provides a delivery system which utilises aretroviral vector.

DETAILED DESCRIPTION

The retroviral vector of the delivery system of the present inventioncomprises a functional splice donor site (“FSDS”) and a functionalsplice acceptor site (“FSAS”) which flank a first NOI. The retroviralvector is formed as a result of reverse transcription of a retroviralpro-vector which may comprise a plurality of NOIs.

When the FSDS is positioned upstream of the FSAS, any interveningsequence(s) are capable of being spliced. Typically, splicing removesintervening or “intronic” RNA sequences and the remaining “exonic”sequences are ligated to provide continuous sequences for translation.

The splicing process can be pictorially represented as:

In this pictorial representation, Y represents the intervening sequencethat is removed as a result of splicing.

The natural splicing configuration for retroviral vectors is shown inFIG. 27a. The splicing configuration of known vectors is shown in FIG.27b. The Splicing configuration according to the present invention isshown in FIG. 27c.

In accordance with the present invention, if the FSDS is downstream ofthe FSAS, then splicing cannot occur.

Likewise, if the FSDS is a non-functional splice donor site (NFSDS)and/or the FSAS is a non-functional acceptor site (NFAS), then splicingcannot occur.

An example of a NFSDS is a mutated FSDS such that the FSDS can no longerbe recognised by the splicing mechanism.

In accordance with the present invention, each NS can be any suitablenucleotide sequence. For example, each sequence can be independently DNAor RNA—which may be synthetically prepared or may be prepared by use ofrecombinant DNA techniques or may be isolated from natural sources ormay be combinations thereof. The sequence may be a sense sequence or anantisense sequence. There may be a plurality of sequences, which may bedirectly or indirectly joined to each other, or combinations thereof.

In accordance with the present invention, each NOI can be any suitablenucleotide sequence. For example, each sequence can be independently DNAor RNA—which may be synthetically prepared or may be prepared by use ofrecombinant DNA techniques or may be isolated from natural sources ormay be combinations thereof. The sequence may be a sense sequence or anantisense sequence. There may be a plurality of sequences, which may bedirectly or indirectly joined to each other, or combinations thereof.

The first NOI may include any one or more of the following selectablemarkers which have been used successfully in retroviral vectors: thebacterial neomycin and hygromycin phosphotransferase genes which conferresistance to G418 and hygromycin respectively (Palmer et al 1987 ProcNatl Acad Sci 84: 1055-1059; Yang et al 1987 Mol Cell Biol 7:3923-3928); a mutant mouse dihydrofolate reductase gene (dhfr) whichconfers resistance to methotrexate (Miller et al 1985 Mol Cell Biol 5:431437); the bacterial gpt gene which allows cells to grow in mediumcontaining mycophenolic acid, xanthine and aminopterin (Mann et al 1983Cell 33: 153-159); the bacterial hisD gene which allows cells to grow inmedium without histidine but containing histidinol (Danos and Mulligan1988 Proc Natl Acad Sci 85: 6460-6464); the multidrug resistance gene(mdr) which confers resistance to a variety of drugs (Guild et al 1988Proc Natl Acad Sci 85: 1595-1599; Pastan et al 1988 Proc. Natl Acad Sci85: 4486-4490) and the bacterial genes which confer resistance topuromycin or phleomycin (Morgenstern and Land 1990 Nucleic Acid Res 18:3587-3596).

All of these markers are dominant selectable markers and allow chemicalselection of most cells expressing these genes. β-galactosidase can alsobe considered a dominant marker; cells expressing β-galactosidase can beselected by using the fluorescence-activated cell sorter. In fact, anycell surface protein can provide a selectable marker for cells notalready making the protein. Cells expressing the protein can be selectedby using the fluorescent antibody to the protein and a cell sorter.Other selectable markers that have been included in vectors include thehprt and HSV thymidine kinase which allows cells to crow in mediumcontaining hypoxanthine, amethopterin and thymidine.

The first NOI could contain non-coding sequences, for example theretroviral packaging site or non-sense sequences that render the secondNOI non-functional in the provector but when they are removed by thesplicing the vector the second NOI is revealed for functionalexpression.

The first NOI may also encode a viral essential element such as envencoding the Env protein which can reduce the complexity of productionsystems. By way of example, in an adenoviral vector, this allows theretroviral vector genome and the envelope to be configured in a singleadenoviral vector under the same promoter control thus providing asimpler system and leaving more capacity in the adenoviral vector foradditional sequences. In one aspect, those additional sequences could bethe gag-pol cassette itself. Thus in one adenoviral vector one canproduce a retroviral vector particle. Previous studies (Feng et al 1997Nature Biotechnology 15: 866) have required the use of multipleadenoviral vectors.

If the retroviral component includes an env nucleotide sequence, thenall or part of that sequence can be optionally replaced with all or partof another env nucleotide sequence such as, by way of example, theamphotropic Env protein designated 4070A or the influenza haemagglutinin(HA) or the vesicular stomatitis virus G (VSV-G) protein. Replacement ofthe env gene with a heterologous env gene is an example of a techniqueor strategy called pseudotyping. Pseudotyping is not a new phenomenonand examples may be found in WO-A-98/05759, WO-A-98105754,WO-A-97/17457, WO-A-96109400, WO-A-91/00047 and Mebatsion et al 1997Cell 90, 841-847.

In one preferred aspect, the retroviral vector of the present inventionhas been pseudotype. In this regard, pseudotyping can confer one or moreadvantages. For example, with the lentiviral vectors, the env geneproduct of the HIV based vectors would restrict these vectors toinfecting only cells that express a protein called CD4. But if the envgene in these vectors has been substituted with env sequences from otherRNA viruses, then they may have a broader infectious spectrum (Verma andSomia 1997 Nature 389:239-242). By way of example, workers havepseudotyped an HIV based vector with the glycoprotein from VSV (Vermaand Somia 1997 ibid).

In another alternative, the Env protein may be a modified Env proteinsuch as a mutant or engineered Env protein. Modifications may be made orselected to introduce targeting ability or to reduce toxicity or foranother purpose (Valsesia-Wittman et al 1996 J Virol 70: 2056-64; Nilsonet al 1996 Gene Therapy 3: 280-6; Fielding et al 1998 Blood 9:1802 andreferences cited therein).

Suitable second NOI coding sequences include those that are oftherapeutic and/or diagnostic application such as, but are not limitedto: sequences encoding cytokines, chemokines, hormones, antibodies,engineered immunoglobulin-like molecules, a single chain antibody,fusion proteins, enzymes, immune co-stimulatory molecules,immunomodulatory molecules, anti-sense RNA, a transdominant negativemutant of a target protein, a toxin, a conditional toxin, an antigen, atumour suppressor protein and growth factors, membrane proteins,vasoactive proteins and peptides, anti-viral proteins and ribozymes, andderivatives therof (such as with an associated reporter group). Whenincluded, such coding sequences may be typically operatively linked to asuitable promoter, which may be a promoter driving expression of aribozyme(s), or a different promoter or promoters.

The second NOI coding sequence may encode a fusion protein or a segmentof a coding sequence

The retroviral vector of the present invention may be used to deliver asecond NOI such as a pro drug activating enzyme to a tumour site for thetreatment of a cancer. In each case, a suitable pro-drug is used in thetreatment of the individual (such as a patient) in combination with theappropriate pro-drug activating enzyme. An appropriate pro-drug isadministered in conjunction with the vector. Examples of pro-drugsinclude: etoposide phosphate (with alkaline phosphatase, Senter et al1988 Proc Natl Acad Sci 85: 48424846); 5-fluorocytosine (with cytosinedeaminase, Mullen et al 1994 Cancer Res 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerret al 1990 Cancer Immunol Immunother 31: 202-206);Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with β-lactanase);SRP4233 (with P450 Reducase); Ganciclovir (with HSV thymidine kinase,Borrelli et al 1988 Proc Natl Acad Sci 85: 7572-7576); mustard pro-drugswith nitroreductase (Friedlos et al 1997 J Med Chem 40: 1270-1275) andCyclophosphamide (with P450 Chen et at 1996 Cancer Res 56: 1331-1340).

The vector of the present invention may be a delivered to a target siteby a viral or a non-viral vector.

As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Byway of example, some vectors used in recombinant DNA techniques allowentities, such as a segment of DNA (such as a heterologous DNA segment,such as a heterologous cDNA segment), to be transferred into a targetcell. Optionally, once within the target cell, the vector may then serveto maintain the heterologous DNA within the cell or may act as a unit ofDNA replication. Examples of vectors used in recombinant DNA techniquesinclude plasmids, chromosomes, artificial chromosomes or viruses.

Non-viral delivery systems include but are not limited to DNAtransfection methods. Here, transfection includes a process using anon-viral vector to deliver a gene to a target mammalian cell.

Typical transfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556),and combinations thereof.

Viral delivery systems include but are not limited to adenovirus vector,an adeno-associated viral (AAV) vector, a herpes viral vector,retroviral vector, lentiviral vector, baculoviral vector. Other examplesof vectors include ex vivo delivery systems, which include but are notlimited to DNA transfection methods such as electroporation, DNAbiolistics, lipid-mediated transfection, compacted DNA-mediatedtransfection.

The vector delivery system of the present invention may consist of aprimary vector manufactured in vitro which encodes the genes necessaryto produce a secondary vector in vivo.

The primary viral vector or vectors may be a variety of different viralvectors, such as retroviral, adenoviral, herpes virus or pox virusvectors, or in the case of multiple primary viral vectors, they may be amixture of vectors of different viral origin. In whichever case, theprimary viral vectors are preferably defective in that they areincapable of independent replication. Thus, they are capable of enteringa target cell and delivering the secondary vector sequences, but not ofreplicating so as to go on to infect further target cells.

In the case where the hybrid viral vector system comprises more than oneprimary vector to encode the secondary vector, both or all three primaryvectors will be used to transfect or transduce a primary target cellpopulation, usually simultaneously.

Preferably, there is a single primary viral vector which encodes allcomponents of the secondary viral vector.

The preferred single or multiple primary viral vectors are adenoviralvectors.

Adenoviral vectors for use in the invention may be derived from a humanadenovirus or an adenovirus which does not normally infect humans.Preferably the vectors are derived from adenovirus type 2 or adenovirustype 5 (Ad2 or Ad5) or a mouse adenovirus or an avian adenovirus such asCELO virus (Cotton et al 1993 J Virol 67:3777-3785). The vectors may bereplication competent adenoviral vectors but are more preferablydefective adenoviral vectors. Adenoviral vectors may be rendereddefective by deletion of one or more components necessary forreplication of the virus. Typically, each adenoviral vector contains atleast a deletion in the E1 region. For production of infectiousadenoviral vector particles, this deletion may be complemented bypassage of the virus in a human embryo fibroblast cell line such ashuman 293 cell line, containing an integrated copy of the left portionof Ad5, including the E1 gene. The capacity for insertion ofheterologous DNA into such vectors can be up to approximately 7 kb. Thussuch vectors are useful for construction of a system according to theinvention comprising three separate recombinant vectors each containingone of the essential transcription units for construction of theretroviral secondary vector.

Alternative adenoviral vectors are known in the art which containfurther deletions in other adenoviral genes and these vectors are alsosuitable for use in the invention. Several of these second generationadenoviral vectors show reduced immunogenicity (eg E1+E2 deletionsGorziglia et al 1996 J Virol 70: 4173-4178; E1+E4 deletions Yeh et al1996 J Virol 70: 559-565). Extended deletions serve to provideadditional cloning capacity for the introduction of multiple genes inthe vector. For example a 25 kb deletion has been described (Lieber etal 1996 3 Virol 70: 8944-8960) and a cloning vector deleted of all viralgenes has been reported (Fisher et al 1996 Virolology 217: 11-22) whichpermit the introduction of more than 35 kb of heterologous DNA. Suchvectors may be used to generate an adenoviral primary vector accordingto the invention encoding two or three transcription units forconstruction of the retroviral secondary vector.

The secondary viral vector is preferably a retroviral vector. Thesecondary vector is produced by expression of essential genes forassembly and packaging of a defective viral vector particle, within theprimary target cells. It is defective in that it is incapable ofindependent replication. Thus, once the secondary retroviral vector hastransduced a secondary target cell, it is incapable of spreading byreplication to any further target cells.

The tern “retroviral vector” typically includes a retroviral nucleicacid which is capable of infection, but which is not capable, by itself,of replication. Thus it is replication defective. A retroviral vectortypically comprises one or more NOI(s), preferably of non-retroviralorigin, for delivery to target cells. A retroviral vector may alsocomprises a functional splice donor site (FSDS) and a functional spliceacceptor site (FSAS) so that when the FSDS is upstream of the FSAS, anyintervening sequence(s) are capable of being spliced. A retroviralvector may comprise further non-retroviral sequences, such asnon-retroviral control sequences in the U3 region which may influenceexpression of an NOI(s) once the retroviral vector is integrated as aprovirus into a target cell. The retroviral vector need not containelements from only a single retrovirus. Thus, in accordance with thepresent invention, it is possible to have elements derivable from two ofmore different retroviruses or other sources

The term “retroviral pro-vector” typically includes a retroviral vectorgenome as described above but which comprises a first nucleotidesequence (NS) capable of yielding a functional splice donor site (FSDs)and a second NS capable of yielding a functional splice acceptor site(FSAS) such that the first NS is downstream of the second NS so thatsplicing associated with the first NS and the second NS cannot occur.Upon reverse transcription of the retroviral pro-vector, a retroviralvector is formed.

The term “retroviral vector particle” refers to the packaged retroviralvector, that is preferably capable of binding to and entering targetcells. The components of the particle, as already discussed for thevector, may be modified with respect to the wild type retrovirus. Forexample, the Env proteins in the proteinaceous coat of the particle maybe genetically modified in order to alter their targeting specificity orachieve some other desired function.

The retroviral vector of this aspect of the invention may be derivablefrom a murine oncoretrovirus such as MMLV, MSV or MMTV; or may bederivable from a lentivirus such as HIV-1, EIAV; or may be derivablefrom another retrovirus.

The retroviral vector of the invention can be modified to render thenatural splice donor site of the retrovirus non-functional.

The term “modification” includes the silencing or removal of the naturalsplice donor. Vectors, such as MLV based vectors, which have the splicedonor site removed are known in the art. An example of such a vector ispBABE (Morgenstern et al 1990 ibid).

The secondary vector may be produced from expression of essential genesfor retroviral vector production encoded in the DNA of the primaryvector. Such genes may include a gag-pol gene from a retrovirus, an envgene from an enveloped virus and a defective retroviral vectorcontaining one or more therapeutic or diagnostic NOI(s). The defectiveretroviral vector contains in general terms sequences to enable reversetranscription, at least part of a 5′ long terminal repeat (LTR), atleast part of a 3′LTR and a packaging signal.

If it is desired to render the secondary vector replication defective,that secondary vector may be encoded by a plurality of transcriptionunits, which may be located in a single or in two or more adenoviral orother primary vectors. Thus, there may be a transcription unit encodingthe secondary vector genome, a transcription unit encoding gag-pol and atranscription unit encoding env. Alternatively, two or more of these maybe combined. For example, nucleic acid sequences encoding gag-pol andenv, or env and the genome, may be combined in a single transcriptionunit. Ways of achieving this are known in the art.

Transcription units as described herein are regions of nucleic acidcontaining coding sequences and the signals for achieving expression ofthose coding sequences independently of any other coding sequences.Thus, each transcription unit generally comprises at least a promoter,an enhancer and a polyadenylation signal.

The term “promoter” is used in the normal sense of the art, e.g. an RNApolymerase binding site in the Jacob-Monod theory of gene expression.

The term “enhancer” includes a DNA sequence which binds to other proteincomponents of the transcription initiation complex and thus facilitatesthe initiation of transcription directed by its associated promoter.

The promoter and enhancer of the transcription units encoding thesecondary vector are preferably strongly active, or capable of beingstrongly induced, in the primary target cells under conditions forproduction of the secondary viral vector. The promoter and/or enhancermay be constitutively efficient, or may be tissue or temporallyrestricted in their activity. Examples of suitable tissue restrictedpromoters/enhancers are those which are highly active in tumour cellssuch as a promoter/enhancer from a MUC1 gene, a CEA gene or a 5T4antigen gene. Examples of temporally restricted promoters/enhancers arethose which are responsive to ischaemia and/or hypoxia, such as hypoxiaresponse elements or the promoter/enhancer of a grp78 or a grp94 gene.One preferred promoter-enhancer combination is a human cytomegalovirus(hCMV) major immediate early (MIE) promoter/enhancer combination.

Other preferred additional components include entities enablingefficient expression of an NOI or a plurality of NOIs.

In one preferred aspect of the present invention, there is hypoxia orischaemia regulatable expression of the secondary vector components. Inthis regard, hypoxia is a powerful regulator of gene expression in awide range of different cell types and acts by the induction of theactivity of hypoxia-inducible transcription factors such as hypoxiainducible factor-1 (HIF-1; Wang & Semenza 1993 Proc Natl Acad Sci90:430), which bind to cognate DNA recognition sites, thehypoxia-responsive elements (HREs) on various gene promoters. Dachs etal (1997 Nature Med 5: 515) have used a multimeric form of the HRE fromthe mouse phosphoglycerate kinase-1 (PGK-1) gene (Firth et al 1994 ProcNatl Acad Sci 91:6496-6500) to control expression of both marker andtherapeutic genes by human fibrosarcoma cells in response to hypoxia invitro and within solid tumours in vivo (Dachs et al ibid).Alternatively, the fact that marked glucose deprivation is also presentin ischaemic areas of tumours can be used to activate heterologous geneexpression specifically in tumours. A truncated 632 base pair sequenceof the grp 78 gene promoter, known to be activated specifically byglucose deprivation, has also been shown to be capable of driving highlevel expression of a reporter gene in murine tumours in vivo (Gazit etal 1995 Cancer Res 55:1660).

An alternative method of regulating the expression of such components isby using the tetracycline on/off system described by Gossen and Bujard(1992 Proc Nat; Acad Sci 89: 5547) as described for the production ofretroviral gal, pol and VSV-G proteins by Yoshida et al (1997 BiochemBiophys Res Comm 230: 426). Unusually this regulatory system is alsoused in the present invention to control the production of thepro-vector genome. This ensures that no vector components are expressedfrom the adenoviral vector in the absence of tetracycline.

Safety features which may be incorporated into the hybrid viral vectorsystem are described below. One or more such features may be present.

The secondary vector is also advantageous for in vivo use in thatincorporated into it are one or more features which eliminate thepossibility of recombination to produce an infectious virus capable ofindependent replication. Such features were not included in previouspublished studies (Feng et al 1997 ibid). In particular, theconstruction of a retroviral vector from three components as describedbelow was not described by Feng et al (ibid).

Firstly, sequence homology between the sequences encoding the componentsof the secondary vector may be avoided by deletion of regions ofhomology. Regions of homology allow genetic recombination to occur. In aparticular embodiment, three transcription units are used to construct asecondary retroviral vector. The first transcription unit contains aretroviral gag-pol gene under the control of a non-retroviral promoterand enhancer. The second transcription unit contains a retroviral envgene under the control of a non-retroviral promoter and enhancer. Thethird transcription unit comprises a defective retroviral genome underthe control of a non-retroviral promoter and enhancer. In the nativeretroviral genome, the packaging signal is located such that part of thegag sequence is required for proper functioning. Normally whenretroviral vector systems are constructed therefrom, the packagingsignal, including part of the gag gene, remains in the vector genome. Inthe present case however, the defective retroviral genome contains aminimal packaging signal which does not contain sequences homologous togag sequences in the first transcription unit. Also, in retroviruses,for example Moloney Murine Leukaemia virus (MMLV), there is a smallregion of overlap between the 3′ end of the pol coding sequence and the5′ end of env. The corresponding region of homology between the firstand second transcription units may be removed by altering the sequenceof either the 3′ end of the pol coding sequence or the 5′ end of env soas to change the codon usage but not the amino acid sequence of theencoded proteins.

Secondly, the possibility of replication competent secondary viralvectors may be avoided by pseudotyping the genome of one retrovirus withthe Env protein of another retrovirus or another enveloped virus so thatregions of homology between the env and gag-pol components are avoided.

In a particular embodiment the retroviral vector is constructed from thefollowing three components: The first transcription unit contains aretroviral gag-pol gene under the control of a non-retroviral promoterand enhancer. The second transcription unit contains the env gene fromthe alternative enveloped virus, under the control of a non-retroviralpromoter and enhancer. The third transcription unit comprises adefective retroviral genome under the control of a non-retroviralpromoter and enhancer. The defective retroviral genome contains aminimal packaging signal which does not contain sequences homologous togag sequences in the first transcription unit.

Thirdly, the possibility of replication competent retroviruses can beeliminated by using two transcription units constructed in a particularway. The first transcription unit contains a gag-pol coding region underthe control of a promoter-enhancer active in the primary target cellsuch as a hCMV promoter-enhancer or a tissue restrictedpromoter-enhancer. The second transcription unit encodes a retroviralgenome RNA capable of being packaged into a retroviral particle. Thesecond transcription unit contains retroviral sequences necessary forpackaging, integration and reverse transcription and also containssequences coding for an env protein of an enveloped virus and the codingsequence of one or more therapeutic genes.

In this example, the transcription of the env and an NOI codingsequences is devised such that the Env protein is preferentiallyproduced in the primary target cell while the NOI expression product isor are preferentially produced in the secondary target cell.

A suitable intron splicing arrangement is described later on in Example5 and illustrated in FIG. 17 and FIG. 27c. Here, a splice donor site ispositioned downstream of a splice acceptor site in the retroviral genomesequence delivered by the primary vector to the primary target cell.Splicing will therefore be absent or infrequent in the primary targetcell so the Env protein will preferentially be expressed. However, oncethe vector genome has gone through the process of reverse transcriptionand integration into the secondary target cell, a functional splicedonor sequence will be located in the 5′ LTR, upstream of a functionalsplice acceptor sequence. Splicing occurs to splice out the env sequenceand transcripts of the NOI are produced.

In a second arrangement of this example, the expression of an NOI isrestricted to the secondary target cell and prevented from beingexpressed in the primary target cell as follows: This arrangement isdescribed later on in Example 6 and illustrated in FIG. 18. There, apromoter-enhancer and a first fragment of an NOI containing the 5′ endof the coding sequence and a natural or artificially derived orderivable splice donor sequence are inserted at the 3′ end of theretroviral genome construct upstream of the R-region. A second fragmentof the NOI which contains all the sequences required to complete thecoding region is placed downstream of a natural or artificially derivedor derivable splice acceptor sequence located downstream from thepackaging signal in the retroviral genome construct. On reversetranscription and integration of the retroviral genome in the secondarytarget cell, the promoter 5′ fragment of the NOI and the functionalsplice donor sequence are located upstream of the functional spliceacceptor and the 3′ end of the NOI. Transcription from the promoter andsplicing then permit translation of the NOI in the secondary targetcell.

In a preferred embodiment the hybrid viral vector system according tothe invention comprises single or multiple adenoviral primary vectorswhich encodes or encode a retroviral secondary vector.

Preferred embodiments of the present invention described address one ofthe major problems associated with adenoviral and other viral vectors,namely that gene expression from such vectors is transient. Theretroviral particles generated from the primary target cells cantransduce secondary target cells and gene expression in the secondarytarget cells is stably maintained because of the integration of theretroviral vector genome into the host cell genome. The secondary targetcells do not express significant amounts of viral protein antigens andso are less immunogenic than cells transduced with adenoviral vector.

The use of a retroviral vector as the secondary vector is advantageousbecause it allows a degree of cellular discrimination, for instance bypermitting the targeting of rapidly dividing cells. Furthermore,retroviral integration permits the stable expression of therapeuticgenes in the target tissue, including stable expression in proliferatingtarget cells.

The use of the novel retroviral vector design of the present inventionis also advantageous in that gene expression can be limited to a primaryor a secondary target site. In this way, single or multiple NOIs can bepreferentially expressed at a secondary target site and poorly expressedor not expressed at a biologically significant level at a primary targetsite. As a result, the possible toxicity or antigenicity of an NOI maybe avoided.

Preferably, the primary viral vector preferentially transduces a certaincell type or cell types.

More preferably, the primary vector is a targeted vector, that is it hasa tissue tropism which is altered compared to the native virus, so thatthe vector is targeted to particular cells.

The term “targeted vector” is not necessarily linked to the term “targetsite” or target cell″.

“Target site” refers to a site which a vector, whether native ortargeted, is capable of transfecting or transducing.

“Primary target site” refers to a first site which a vector, whethernative or targeted, is capable of transfecting or transducing.

“Secondary target site” refers to a second site which a vector, whethernative or targeted, is capable of transfecting or transducing.

“Target cell” simply refers to a cell which a vector, whether native ortargeted, is capable of transfecting or transducing.

“Primary target cell” refers to a first cell which a vector, whethernative or targeted, is capable of transfecting or transducing.

“Secondary target cell” refers to a second cell which a vector, whethernative or targeted, is capable of transfecting or transducing.

The preferred, adenoviral primary vector according to the invention isalso preferably a targeted vector, in which the tissue tropism of thevector is altered from that of a wild-type adenovirus. Adenoviralvectors can be modified to produce targeted adenoviral vectors forexample as described in: Krasnykh et al 1996 J. Virol 70: 6839-6846;Wickham et al 1996 J. Virol 70: 6831-838; Stevenson et al 1997 J. Virol71: 4782-4790; Wickham et at 1995 Gene Therapy 2: 750-756; Douglas et at1997 Neuromuscul. Disord 7:284-298; Wickham et at 1996 NatureBiotechnology 14: 1570-1573.

Primary target cells for the vector system according to the inventioninclude s haematopoietic cells (including monocytes, macrophages,lymphocytes, granulocytes or progenitor cells of any of these);endothelial cells; tumour cells; stromal cells; astrocytes or glialcells; muscle cells; and epithelial cells.

Thus, a primary target cell according to the invention, capable ofproducing the second viral vector, may be of any of the above celltypes.

In a preferred embodiment, the primary target cell according to theinvention is a monocyte or macrophage transduced by a defectiveadenoviral vector containing a first transcription unit for a retroviralgag-pol and a second transcription unit capable of producing apackageable defective retroviral genome. In this case at least thesecond transcription unit is preferably under the control of apromoter-enhancer which is preferentially active in a diseased locationwithin the body such as an ischaemic site or the micro-environment of asolid tumour.

In a particularly preferred embodiment, the second transcription unit isconstructed such that on insertion of the genome into the secondarytarget cell, an intron is generated which serves to reduce expression ofa viral essential element, such as the viral env gene, and permitefficient expression of a therapeutic and/or diagnostic NOI or NOIs.

The packaging cell may be an in vivo packaging cell in the body of anindividual to be treated or it may be a cell cultured in vitro such as atissue culture cell line. Suitable cell lines include mammalian cellssuch as murine fibroblast derived cell lines or human cell lines.Preferably the packaging cell line is a human cell line, such as forexample: HEK293, 293-T, TE671, HT1080.

Alternatively, the packaging cell may be a cell derived from theindividual to be treated such as a monocyte, macrophage, blood cell orfibroblast. The cell may be isolated from an individual and thepackaging and vector components administered ex vivo followed byre-administration of the autologous packaging cells. Alternatively thepackaging and vector components may be administered to the packagingcell in vivo. Methods for introducing retroviral packaging and vectorcomponents into cells of an individual are known in the art. Forexample, one approach is to introduce the different DNA sequences thatare required to produce a retroviral vector particle e.g. the env codingsequence, the gag-pol coding sequence and the defective retroviralgenome into the cell simultaneously by transient triple transfection(Landau & Littman 1992 J. Virol. 66, 5110; Soneoka et al 1995 NucleicAcids Res 23:628-633).

The secondary viral vectors may also be targeted vectors. For retroviralvectors, this may be achieved by modifying the Env protein. The Envprotein of the retroviral secondary vector needs to be a non-toxicenvelope or an envelope which may be produced in non-toxic amountswithin the primary target cell, such as for example a MMLV amphotropicenvelope or a modified amphotropic envelope. The safety feature in sucha case is preferably the deletion of regions or sequence homologybetween retroviral components.

Preferably the envelope is one which allows transduction of human cells.Examples of suitable env genes include, but are not limited to, VSV-G, aMLV amphotropic env such as the 4070A env, the RD114 feline leukaemiavirus env or haemagglutinin (HA) from an influenza virus. The Envprotein may be one which is capable of binding to a receptor on alimited number of human cell types and may be an engineered envelopecontaining targeting moieties. The env and gag-pol coding sequences aretranscribed from a promoter and optionally an enhancer active in thechosen packaging cell line and the transcription unit is terminated by apolyadenylation signal. For example, if the packaging cell is a humancell, a suitable promoter-enhancer combination is that from the humancytomegalovirus major immediate early (hCMV-MIE) gene and apolyadenylation signal from SV40 virus may be used. Other suitablepromoters and polyadenylation signals are known in the art.

The secondary target cell population may be the same as the primarytarget cell population. For example delivery of a primary vector of theinvention to tumour cells leads to replication and generation of furthervector particles which can transduce further tumour cells.

Alternatively, the secondary target cell population may be differentfrom the primary target cell population. In this case the primary targetcells serve as an endogenous factory within the body of the treatedindividual and produce additional vector particles which can transducethe secondary target cell population. For example, the primary targetcell population may be haematopoietic cells transduced by the primaryvector in vivo or ea vivo. The primary target cells are then deliveredto or migrate to a site within the body such as a tumour and produce thesecondary vector particles, which are capable of transducing for examplemitotically active tumour cells within a solid tumour.

The retroviral vector particle according to the invention will also becapable of transducing cells which are slowly-dividing, and whichnon-lentiviruses such as MLV would not be able to efficiently transduce.Slowly-dividing cells divide once in about every three to four daysincluding certain tumour cells. Although tumours contain rapidlydividing cells, some tumour cells especially those in the centre of thetumour, divide infrequently. Alternatively the target cell may be agrowth-arrested cell capable of undergoing cell division such as a cellin a central portion of a tumour mass or a stem cell such as ahaematopoietic stem cell or a CD34-positive cell. As a furtheralternative, the target cell may be a precursor of a differentiated cellsuch as a monocyte precursor, a CD33-positive cell, or a myeloidprecursor. As a further alternative, the target cell may be adifferentiated cell such as a neuron, astrocyte, glial cell, microglialcell, macrophage, monocyte, epithelial cell, endothelial cell,hepatocyte, spermatocyte, spermatid or spermatozoa. Target cells may betransduced either in vitro after isolation from a human individual ormay be transduced directly in vivo.

The invention permits the localised production of high titres ofdefective retroviral vector particles in vivo at or near the site atwhich action of a therapeutic protein or proteins is required withconsequent efficient transduction of secondary target cells. This ismore efficient than using either a defective adenoviral vector or adefective retroviral vector alone.

The invention also permits the production of retroviral vectors such asMMLV-based vectors in non-dividing and slowly-dividing cells in vivo. Ithad previously been possible to produce MMLV-based retroviral vectorsonly in rapidly dividing cells such as tissue culture-adapted cellsproliferating in vitro or rapidly dividing tumour cells in vivo.Extending the range of cell types capable of producing retroviralvectors is advantageous for delivery of genes to the cells of solidtumours, many of which are dividing slowly, and for the use ofnon-dividing cells such as endothelial cells and cells of varioushaematopoietic lineages as endogenous factories for the production oftherapeutic protein products.

The delivery of one or more therapeutic genes by a vector systemaccording to the present invention may be used alone or in combinationwith other treatments or components of the treatment.

For example, the retroviral vector of the present invention may be usedto deliver one or more NOI(s) useful in the treatment of the disorderslisted in WO-A-98/05635. For ease of reference, part of that list is nowprovided: cancer, inflammation or inflammatory disease, dermatologicaldisorders, fever, cardiovascular effects, haemorrhage, coagulation andacute phase response, cacbexia, anorexia, acute infection, HIVinfection, shock states, graft-versus-host reactions, autoimmunedisease, reperfusion injury, meningitis, migraine and aspirin-dependentanti-thrombosis; tumour growth, invasion and spread, angiogenesis,metastases, malignant, ascites and malignant pleural effusion; cerebralischaemia, ischaemic heart disease, osteoarthritis, rheumatoidarthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration,Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn'sdisease and ulcerative colitis; periodontitis, gingivitis; psoriasis,atopic dermatitis, chronic ulcers, epidermolysis bullosa; cornealulceration, retinopathy and surgical wound healing; rhinitis, allergicconjunctivitis, eczema, anaphylaxis; restenosis, congestive heartfailure, endometriosis, atherosclerosis or endosclerosis.

In addition, or in the alternative, the retroviral vector of the presentinvention may be used to deliver one or more NOI(s) useful in thetreatment of disorders listed in WO-A-98/07859. For ease of reference,part of that list is now provided: cytokine and cellproliferation/differentiation activity; immunosuppressant orimmunostimulant activity (e.g. for treating immune deficiency, includinginfection with human immune deficiency virus; regulation of lymphocytegrowth; treating cancer and many autoimmune diseases, and to preventtransplant rejection or induce tumour immunity); regulation ofhaematopoiesis, e.g. treatment of myeloid or lymphoid diseases;promoting growth of bone, cartilage, tendon, ligament and nerve tissue,e.g. for healing wounds, treatment of burns, ulcers and periodontaldisease and neurodegeneration; inhibition or activation offollicle-stimulating hormone (modulation of fertility);chemotactic/chemokinetic activity (e.g. for mobilising specific celltypes to sites of injury or infection); haemostatic and thrombolyticactivity (e.g. for treating haemophilia and stroke); antiinflammatoryactivity (for treating e.g. septic shock or Crohn's disease); asantimicrobials; modulators of e.g. metabolism or behaviour; asanalgesics; treating specific deficiency disorders; in treatment of e.g.psoriasis, in human or veterinary medicine.

In addition, or in the alternative, the retroviral vector of the presentinvention may be used to deliver one or more NOI(s) useful in thetreatment of disorders listed in WO-A-98/09985. For ease of reference,part of that list is now provided: macrophage inhibitory and/or T cellinhibitory activity and thus, anti-inflammatory activity; anti-immuneactivity, i.e. inhibitory effects against a cellular and/or humoralimmune response, including a response not associated with inflammation;inhibit the ability of macrophages and T cells to adhere toextracellular matrix components and fibronectin, as well as up-regulatedfas receptor expression in T cells; inhibit unwanted immune reaction andinflammation including arthritis, including rheumatoid arthritis,inflammation associated with hypersensitivity, allergic reactions,asthma, systemic lupus erythematosus, collagen diseases and otherautoimmune diseases, inflammation associated with atherosclerosis,arteriosclerosis, atherosclerotic heart disease, reperfusion injury,cardiac arrest, myocardial infarction, vascular inflammatory disorders,respiratory distress syndrome or other cardiopulmonary diseases,inflammation associated with peptic ulcer, ulcerative colitis and otherdiseases of the gastrointestinal tract, hepatic fibrosis, livercirrhosis or other hepatic diseases, thyroiditis or other glandulardiseases, glomerulonephritis or other renal and urologic diseases,otitis or other -oto-rhino-laryngological diseases, dermatitis or otherdermal diseases, periodontal diseases or other dental diseases, orchitisor epididimo-orchitis, infertility, orchidal trauma or otherimmune-related testicular diseases, placental dysfunction, placentalinsufficiency, habitual abortion, eclampsia, pre-eclampsia and otherimmune and/or inflammatory-related gynaecological diseases, posterioruveitis, intermediate uveitis, anterior uveitis, conjunctivitis,chorioretinitis, uveoretinitis, optic neuritis, intraocularinflammation, e.g. retinitis or cystoid macular oedema, sympatheticophthalmia, scleritis, retinitis pigmentosa, immune and inflammatorycomponents of degenerative fondus disease, inflammatory components ofocular trauma, ocular inflammation caused by infection, proliferativevitreo-retinopathies, acute ischaemic optic neuropathy, excessivescarring, e.g. following glaucoma filtration operation, immune and/orinflammation reaction against ocular implants and other immune andinflammatory-related ophthalmic diseases, inflammation associated withautoimmune diseases or conditions or disorders where, both in thecentral nervous system (CNS) or in any other organ, immune and/orinflammation suppression would be beneficial, Parkinson's disease,complication and/or side effects from treatment of Parkinson's disease,AIDS-related dementia complex HIV-related encephalopathy, Devic'sdisease, Sydenham chorea, Alzheimer's disease and other degenerativediseases, conditions or disorders of the CNS, inflammatory components ofstokes, post-polio syndrome, immune and inflammatory components ofpsychiatric disorders, myelitis, encephalitis, subacute sclerosingpan-encephalitis, encephalomyelitis, acute neuropathy, subacuteneuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora,myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington'sdisease, amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.

Further provided according to the invention are methods of controllingproduction of a therapeutic NOI or NOIs such that the therapeutic NOI orNOIs is/are preferentially expressed in a secondary target cellpopulation and is/are poorly expressed or not expressed at abiologically significant level in a primary target cell.

The present invention also provides a pharmaceutical composition fortreating an individual by gene therapy, wherein the compositioncomprises a therapeutically effective amount of the retroviral vector ofthe present invention comprising one or more deliverable therapeuticand//or diagnostic NOI(s) or a viral particle produced by or obtainedfrom same. The pharmaceutical composition may be for human or animalusage. Typically, a physician will determine the actual dosage whichwill be most suitable for an individual subject and it will vary withthe age, weight and response of the particular individual.

The composition may optionally comprise a pharmaceutically acceptablecarrier, diluent, excipient or adjuvant. The choice of pharmaceuticalcarrier, excipient or diluent can be selected with regard to theintended route of administration and standard pharmaceutical practice.The pharmaceutical compositions may comprise as—or in addition to—thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s), and othercarrier agents that may aid or increase the viral entry into the targetsite (such as for example a lipid delivery system).

Where appropriate, the pharmaceutical compositions can be administeredby any one or more of: inhalation, in the form of a suppository orpessary, topically in the form of a lotion, solution, cream, ointment ordusting powder, by use of a skin patch, orally in the form of tabletscontaining excipients such as starch or lactose, or in capsules orovules either alone or in admixture with excipients, or in the form ofelixirs, solutions or suspensions containing flavouring or colouringagents, or they can be injected parenterally, for exampleintracavernosally, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions may be best used in the formof a sterile aqueous solution which may contain other substances, forexample enough salts or monosaccharides to make the solution isotonicwith blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can beformulated in a conventional manner.

In a further aspect of the present invention, there is provided a hybridviral vector system in the general sense (i.e. not necessarily limitedto the aforementioned first aspect of the present invention as definedabove) for in vivo gene delivery, which system comprises one or moreprimary viral vectors which encode a secondary viral vector, the primaryvector or vectors capable of infecting a first target cell and ofexpressing therein the secondary viral vector, which secondary vector iscapable of transducing a secondary target cell.

With this particular embodiment, the genetic vector of the invention isthus a hybrid viral vector system for gene delivery which is capable ofgeneration of defective infectious particles from within a target cell.Thus a genetic vector of the invention consists of a primary vectormanufactured in vitro which encodes the genes necessary to produce asecondary vector in viva. In use, the secondary vector carries one ormore selected genes for insertion into the secondary target cell. Theselected genes may be one or more marker genes and/or therapeutic genes.Marker genes encode selectable and/or detectable proteins.

More aspects concerning this particular aspect of the present inventionnow follow—which teachings are also applicable to the aforementionedaspects of the present invention.

In another aspect the invention provides target cells infected by theprimary viral vector or vectors and capable of producing infectioussecondary viral vector particles.

In a further aspect the invention provides a method of treatment of ahuman or non-human mammal, which method comprises administering a hybridviral vector system or target cells infected by the primary viral vectoror vectors, as described herein.

The primary viral vector or vectors may be a variety of different viralvectors, such as retroviral, adenoviral, herpes virus or pox virusvectors, or in the case of multiple primary viral vectors, they may be amixture of vectors of different viral origin. In whichever case, theprimary viral vectors are preferably defective in that they areincapable of independent replication. Thus, they are capable of enteringa target cell and delivering the secondary vector sequences, but not ofreplicating so as to go on to infect further target cells.

In the case where the hybrid viral vector system comprises more than oneprimary vector to encode the secondary vector, both or all three primaryvectors will be used to infect a primary target cell population, usuallysimultaneously. Preferably, there is a single primary viral vector whichencodes all components of the secondary viral vector.

The preferred single or multiple primary viral vectors are adenoviralvectors. Adenovirus vectors have significant advantages over other viralvectors in terms of the titres which can be obtained from in vitrocultures. The adenoviral particles are also comparatively stablecompared with those of enveloped viruses and are therefore more readilypurified and stored. However, current adenoviral vectors suffer frommajor limitations for in vivo therapeutic use since gene expression fromdefective adenoviral vectors is only transient.

Because the vector genome does not replicate, target cell proliferationleads to dilution of the vector. Also cells expressing adenoviralproteins, even at a low level, are destroyed by an immunologicalresponse raised against the adenoviral proteins.

The secondary viral vector is preferably a retroviral vector. Thesecondary vector is produced by expression of essential genes forassembly and packaging of a defective viral vector particle, within theprimary target cells. It is defective in that it is incapable ofindependent replication. Thus, once the secondary retroviral vector hastransduced a secondary target cell, it is incapable of spreading byreplication to any further target cells.

The secondary vector may be produced from expression of essential genesfor retroviral vector production encoded in the DNA of the primaryvector. Such genes may include a gag-pol gene from a retrovirus, anenvelope gene from an enveloped virus and a defective retroviral genomecontaining one or more therapeutic genes. The defective retroviralgenome contains in general terms sequences to enable reversetranscription, at least part of a 5′ long terminal repeat (LTR), atleast part of a 3′LTR and a packaging signal.

Importantly, the secondary vector is also safe for in vivo use in thatincorporated into it are one or more safety features which eliminate thepossibility of recombination to produce an infectious virus capable ofindependent replication.

To ensure that it is replication defective the secondary vector may beencoded by a plurality of transcription units, which may be located in asingle or in two or more adenoviral or other primary vectors. Thus,there may be a transcription unit encoding the secondary vector genome,a transcription unit encoding gag-pol and a transcription unit encodingenv. Alternatively, two or more of these may be combined. For example,nucleic acid sequences encoding gag-pol and env, or env and the genome,may be combined in a single transcription unit. Ways of achieving thisare known in the art.

Transcription units as described herein are regions of nucleic acidcontaining coding sequences and the signals for achieving expression ofthose coding sequences independently of any other coding sequences.Thus, each transcription unit generally comprises at least a promoter,an enhancer and a polyadenylation signal. The promoter and enhancer ofthe transcription units encoding the secondary vector are preferablystrongly active, or capable of being strongly induced, in the primarytarget cells under conditions for production of the secondary viralvector. The promoter and/or enhancer may be constitutively efficient, ormay be tissue or temporally restricted in their activity. Examples ofsuitable tissue restricted promoters/enhancers are those which arehighly active in tumour cells such as a promoter/enhancer from a MUC 1gene, a CEA gene or a 5T4 antigen gene. Examples of temporallyrestricted promoters/enhancers are those which are responsive toischaemia and/or hypoxia, such as hypoxia response elements or thepromoter/enhancer of a grp78 or a grp94 gene. One preferredpromoter-enhancer combination is a human cytomegalovirus (hCMV) majorimmediate early (MIE) promoter/enhancer combination.

Hypoxia or ischaemia regulatable expression of secondary vectorcomponents may be particularly useful under certain circumstances.Hypoxia is a powerful regulator of gene expression in a wide range ofdifferent cell types and acts by the induction of the activity ofhypoxia-inducible transcription factors such as hypoxia induciblefactor-1 (HIF-1; Wang & Semenza (1993). Proc. Natl. Acad. Sci USA90:430), which bind to cognate DNA recognition sites, thehypoxia-responsive elements (HREs) on various gene promoters. Dachs etal (1997). Nature Med. 5: 515.) have used a multimeric form of the HREfrom the mouse phosphoglycerate kinase-1 (PGK-1) gene (Firth et al.(1994). Proc. Natl. Acad. Sci USA 91:6496-6500) to control expression ofboth marker and therapeutic genes by human fibrosarcoma cells inresponse to hypoxia in vitro and within solid tumours in vivo (Dachs etal ibid). Alternatively, the fact that marked glucose deprivation isalso present in ischaemic areas of tumours can be used to activateheterologous gene expression specifically in tumours. A truncated 632base pair sequence of the grp 78 gene promoter, known to be activatedspecifically by glucose deprivation, has also been shown to be capableof driving high level expression of a reporter gene in murine tumours invivo (Gazit G, et al (1995). Cancer Res. 55:1660).

Safety features which may be incorporated into the hybrid viral vectorsystem are described below. One or more such features may be present.

Firstly, sequence homology between the sequences encoding the componentsof the secondary vector may be avoided by deletion of regions ofhomology. Regions of homology allow genetic recombination to occur. In aparticular embodiment, three transcription units are used to construct asecondary retroviral vector. A first transcription unit contains aretroviral gag-pol gene under the control of a non-retroviral promoterand enhancer. A second transcription unit contains a retroviral env geneunder the control of a non-retroviral promoter and enhancer. A thirdtranscription unit comprises a defective retroviral genome under thecontrol of a non-retroviral promoter and enhancer. In the nativeretroviral genome, the packaging signal is located such that part of thegag sequence is required for proper functioning. Normally whenretroviral vector systems are constructed therefore, the packagingsignal, including part of the gag gene, remains in the vector genome. Inthe present case however, the defective retroviral genome contains aminimal packaging signal which does not contain sequences homologous togag sequences in the first transcription unit. Also, in retroviruses,for example Moloney Murine Leukaemia virus (MMLV), there is a smallregion of overlap between the 3′ end of the pol coding sequence and the5′ end of env. The corresponding region of homology between the firstand second transcription units may be removed by altering the sequenceof either the 3′ end of the pol coding sequence or the 5′ end of env soas to change the codon usage but not the amino acid sequence of theencoded proteins.

Secondly, the possibility of replication competent secondary viralvectors may be avoided by pseudotyping the genome of one retrovirus withthe envelope protein of another retrovirus or another enveloped virus sothat regions of homology between the env and gag-pol components areavoided. In a particular embodiment the retroviral vector is constructedfrom the following three components. The first transcription unitcontains a retroviral gag-pol gene under the control of a non-retroviralpromoter and enhancer. The second transcription unit contains the envgene from the alternative enveloped virus, under the control of anon-retroviral promoter and enhancer. The third transcription unitcomprises a defective retroviral genome under the control of anon-retroviral promoter and enhancer. The defective retroviral genomecontains a minimal packaging signal which does not contain sequenceshomologous to gag sequences in the first transcription unit.

Pseudotyping may involve for example a retroviral genome based on alentivirus such as an HIV or equine infectious anaemia virus (EIAV) andthe envelope protein may for example be the amphotropic envelope proteindesignated 4070A. Alternatively, the retroviral genome may be based onMMLV and the envelope protein may be a protein from another virus whichcan be produced in non-toxic amounts within the primary target cell suchas an Influenza haemagglutinin or vesicular stomatitis virus G protein.In another alternative, the envelope protein may be a modified envelopeprotein such as a mutant or engineered envelope protein. Modificationsmay be made or selected to introduce targeting ability or to reducetoxicity or for another purpose.

Thirdly, the possibility of replication competent retroviruses can beeliminated by using two transcription units constructed in a particularway. The first transcription unit contains a gag-pol coding region underthe control of a promoter-enhancer active in the primary target cellsuch as a hCMV promoter-enhancer or a tissue restrictedpromoter-enhancer. The second transcription unit encodes a retroviralgenome RNA capable of being packaged into a retroviral particle. Thesecond transcription unit contains retroviral sequences necessary forpackaging, integration and reverse transcription and also containssequences coding for an env protein of an enveloped virus and the codingsequence of one or more therapeutic genes.

In a preferred embodiment the hybrid viral vector system according tothe invention comprises single or multiple adenoviral primary vectorswhich encodes or encode a retroviral secondary vector. Adenoviralvectors for use in the invention may be derived from a human adenovirusor an adenovirus which does not normally infect humans. Preferably thevectors are derived from Adenovirus Type 2 or adenovirus Type 5 (Ad2 orAd5) or a mouse adenovirus or an avian adenovirus such as CELO virus(Cotton et al 1993 J. Virol. 67:3777-3785). The vectors may bereplication competent adenoviral vectors but are more preferablydefective adenoviral vectors. Adenoviral vectors may be rendereddefective by deletion of one or more components necessary forreplication of the virus. Typically, each adenoviral vector contains atleast a deletion in the E1 region. For production of infectiousadenoviral vector particles, this deletion may be complemented bypassage of the virus in a human embryo fibroblast cell line such ashuman 293 cell line, containing an integrated copy of the left portionof Ad5, including the E1 gene. The capacity for insertion ofheterologous DNA into such vectors can be up to approximately 7 kb. Thussuch vectors are useful for construction of a system according to theinvention comprising three separate recombinant vectors each containingone of the essential transcription units for construction of theretroviral secondary vector.

Alternative adenoviral vectors are known in the art which containfurther deletions in other adenoviral genes and these vectors are alsosuitable for use in the invention. Several of these second generationadenoviral vectors show reduced immunogenicity (eg E1+E2 deletionsGorziglia et al 1996 J. Virol. 70: 4173-4178; E1+E4 deletions Yeh et al1996 J. Virol. 70: 559-565). Extended deletions serve to provideadditional cloning capacity for the introduction of multiple genes inthe vector. For example a 25 kb deletion has been described (Lieber etal. 1996 J. Virol. 70: 8944-8960) and a cloning vector deleted of allviral genes has been reported (Fisher et al 1996 Virolology 217: 11-22)which will permit the introduction of more than 35 kb of heterologousDNA. Such vectors may be used to generate an adenoviral primary vectoraccording to the invention encoding two or three transcription units forconstruction of the retroviral secondary vector.

Embodiments of the invention described solve one of the major problemsassociated with adenoviral and other viral vectors, namely that geneexpression from such vectors is transient. The retroviral particlesgenerated from the primary target cells can infect secondary targetcells and gene expression in the secondary target cells is stablymaintained because of the integration of the retroviral vector genomeinto the host cell genome. The secondary target cells do not expresssignificant amounts of viral protein antigens and so are lessimmunogenic than the cells transduced with adenoviral vector.

The use of a retroviral vector as the secondary vector is alsoadvantageous because it allows a degree of cellular discrimination, forinstance by permitting the targeting of rapidly dividing cells.Furthermore, retroviral integration permits the stable expression oftherapeutic genes in the target tissue, including stable expression inproliferating target cells.

Preferably, the primary viral vector preferentially infects a certaincell type or cell types. More preferably, the primary vector is atargeted vector, that is it has a tissue tropism which is alteredcompared to the native virus, so that the vector is targeted toparticular cells. The term “targeted vector” is not necessarily linkedto the term “target cell”. “target cell” simply refers to a cell which avector, whether native or targeted, is capable of infecting ortransducing.

The preferred, adenoviral primary vector according to the invention isalso preferably a targeted vector, in which the tissue tropism of thevector is altered from that of a wild-type adenovirus. Adenoviralvectors can be modified to produce targeted adenoviral vectors forexample as described in Krasnykh et al. 1996 J. Virol 70: 6839-6846;Wickham et al 1996 J. Virol 70: 6831-6838; Stevenson et al. 1997 J.Virol. 71: 4782-4790; Wickham et al. 1995 Gene Therapy 2: 750-756;Douglas et al. 1997 Neuromuscul. Disord. 7:284-298; Wickham et al. 1996Nature Biotechnology 14: 1570-1573.

Primary target cells for the vector system according to the inventioninclude but are not limited to haematopoietic cells (includingmonocytes, macrophages, lymphocytes, granulocytes or progenitor cells ofany of these); endothelial cells; tumour cells; stromal cells;astrocytes or glial cells; muscle cells; and epithelial cells.

Thus, a primary target cell according to the invention, capable ofproducing the second viral vector, may be of any of the above celltypes. In a preferred embodiment, the primary target cell according tothe invention is a monocyte or macrophage infected by a defectiveadenoviral vector containing a first transcription unit for a retroviralgag-pol and a second transcription unit capable of producing apackageable defective retroviral genome. In this case at least thesecond transcription unit is preferably under the control of apromoter-enhancer which is preferentially active in a diseased locationwithin the body such as an ischaemic site or the micro-environment of asolid tumour. In a particularly preferred embodiment of this aspect ofthe invention, the second transcription unit is constructed such that oninsertion of the genome into the secondary target cell, an intron isgenerated which serves to reduce expression of the viral env gene andpermit efficient expression of a therapeutic gene.

The secondary viral vectors may also be targeted vectors. For retroviralvectors, this may be achieved by modifying the envelope protein. Theenvelope protein of the retroviral secondary vector needs to be anon-toxic envelope or an envelope which may be produced in non-toxicamounts within the primary target cell, such as for example a MMLVamphotropic envelope or a modified amphotropic envelope. The safetyfeature in such a case is preferably the deletion of regions or sequencehomology between retroviral components.

The secondary target cell population may be the same as the primarytarget cell population. For example delivery of a primary vector of theinvention to tumour cells leads to replication and generation of furthervector particles which can transduce further tumour cells.Alternatively, the secondary target cell population may be differentfrom the primary target cell population. In this case the primary targetcells serve as an endogenous factory within the body of the treatedindividual and produce additional vector particles which can infect thesecondary target cell population. For example, the primary target cellpopulation may be haematopoietic cells transduced by the primary vectorin vivo or ex vivo. The primary target cells are then delivered to ormigrate to a site within the body such as a tumour and produce thesecondary vector particles, which are capable of transducing for exampletumour cells within a solid tumour.

The invention permits the localised production of high titres ofdefective retroviral vector particles in vivo at or near the site atwhich action of a therapeutic protein or proteins is required withconsequent efficient transduction of secondary target cells. This ismore efficient than using either a defective adenoviral vector or adefective retroviral vector alone.

The invention also permits the production of retroviral vectors such asMMLV-based vectors in non-dividing and slowly-dividing cells in vivo. Ithad previously been possible to produce MMLV-based retroviral vectorsonly in rapidly dividing cells such as tissue culture-adapted cellsproliferating in vitro or rapidly dividing tumour cells in vivo.Extending the range of cell types capable of producing retroviralvectors is advantageous for delivery of genes to the cells of solidtumours, many of which are dividing slowly, and for the use of nondividing cells such as endothelial cells and cells of varioushaematopoietic lineages as endogenous factories for the production oftherapeutic protein products.

The delivery of one or more therapeutic genes by a vector systemaccording to the invention may be used alone or in combination withother treatments or components of the treatment. Diseases which may betreated include, but are not limited to: cancer, neurological diseases,inherited diseases, heart disease, stroke, arthritis, viral infectionsand diseases of the immune system. Suitable therapeutic genes includethose coding for tumour suppressor proteins, enzymes, pro-drugactivating enzymes, immunomodulatory molecules, antibodies, engineeredimmunoglobulin-like molecules, fusion proteins, hormones, membraneproteins, vasoactive proteins or peptides, cytokines, chemokines,anti-viral proteins, antisense RNA and ribozymes.

In a preferred embodiment of a method of treatment according to theinvention, a gene encoding a pro-drug activating enzyme is delivered toa tumour using the vector system of the invention and the individual issubsequently treated with an appropriate pro-drug. Examples of pro-drugsinclude etoposide phosphate (used with alkaline phosphatase Senter etal., 1988 Proc. Natl. Acad. Sci. 85: 4842-4846); 5-fluorocytosine (withCytosine deaminase Mullen et al. 1994 Cancer Res. 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase (Kerret al. 1990 Cancer Immunol. Immunother. 31: 202-206);Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with CarboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with b-lactamase);SR4233 (with P450 Reducase); Ganciclovir (with HSV thymidine kinase,Borrelli et al. 1988 Proc . Natl. Acad. Sci. 85: 7572-7576) mustardpro-drugs with nitroreductase (Friedlos et al. 1997J Med Chem 40:1270-1275) and Cyclophosphamide or Ifosfamide (with a cytochrome P450Chen et al. 1996 Cancer Res 56: 1331-1340).

Further provided according to the invention are methods of controllingproduction of a therapeutic gene such that the therapeutic gene ispreferentially expressed in the secondary target cell population and ispoorly expressed or not expressed at a biologically significant level inthe primary target cell.

In accordance with the invention, standard molecular biology techniquesmay be used which are within the level of skill in the art. Suchtechniques are fully described in the literature. See for example;Sambrook et al (1989) Molecular Cloning; a laboratory manual; Hames andGlover (1985-1997) DNA Cloning: a practical approach, Volumes I-IV(second edition); Methods for the engineering of immunoglobulin genesare given in McCafferty et al (1996) “Antibody Engineering: A PracticalApproach”.

In summation, the present invention relates to a novel delivery systemsuitable for introducing one or more NOIs into a target cell.

In one broad aspect the present invention relates to a retroviral vectorcomprising a functional splice donor site and a functional spliceacceptor site; wherein the functional splice donor site and thefunctional splice acceptor site flank a first nucleotide sequence ofinterest (“NOI”); wherein the functional splice donor site is upstreamof the functional splice acceptor site; wherein the retroviral vector isderived from a retroviral pro-vector; wherein the retroviral pro-vectorcomprises a first nucleotide sequence (“NS”) capable of yielding thefunctional splice donor site and a second NS capable of yielding thefunctional splice acceptor site; wherein the first NS is downstream ofthe second NS; such that the retroviral vector is formed as a result ofreverse transcription of the retroviral pro-vector.

In a further broad aspect, the present invention provides a hybrid viralvector system for in vivo gene delivery, which system comprises one ormore primary viral vectors which encode a secondary viral vector, theprimary vector or vectors capable of infecting a first target cell andof expressing therein the secondary viral vector, which secondary vectoris capable of transducing a secondary target cell.

Preferably the primary vector is obtainable from or is based on aadenoviral vector and/or the secondary viral vector is obtainable fromor is based on a retroviral vector preferably a lentiviral vector.

The invention will now be further described by way of example in whichreference is made to the following Figures:

FIG. 1 which shows the structure of a retroviral proviral genome;

FIG. 2 which shows the addition of a small T splice donor pLTR (SEQ IDNOS 23 and 24, respectively, in order of appearance);

FIG. 3 which shows a diagrammatic representation of pL-SA-N (SEQ ID NOS25, and 26, respectively, in order of appearance);

FIG. 4 which shows a diagrammatic representation of pL-SA-N with asplice donor deletion (SEQ ID NOS 27 and 28, respectively, in order ofappearance); FIG. 5 which shows the sequence of MLV pICUT (SEQ ID NO:1);

FIG. 6 which shows the insertion of a splice donor at CMV/R junction ofELAV LTR plasmid (SEQ ID NOS 29 and 30, respectively, in order ofappearance);

FIG. 7 which shows the insertion of a splice acceptor into pEGASUS-1(SEQ ID NOS 31 and 32, respectively, in order of appearance);

FIG. 8 which shows the removal of a wild-type splice donor from EIAVvector (SEQ ID NOS 33-36 respectively, in order of appearance);

FIG. 9 which shows the combination of pCMVLTR+SD with pEGASUS+SA (noSD)to create pEICUT-1;

FIG. 10 which shows the construction of pEICUT-LacZ

FIG. 11 which shows the pEICUT-LacZ sequence (SEQ ID NO: 2);

FIG. 12 which shows the vector configuration in both transfected andtransduced cells;

FIG. 13 which shows the restriction of gene expression to eitherpackaging or transduced cells;

FIG. 14 which shows the construction of a MLV pICUT Neo-p450 vector thatrestricts hygromycin expression to producer cells and 2B6 (a p450isoform) expression to transduced cells;

FIG. 15 which shows a sequence comparison of mutant env (m4070A) (SEQ IDNO: 5) with wild type MMLV sequencer (SEQ ID NO: 4) from the 3′ end ofthe pol gene;

FIG. 16 which shows the complete sequence (SEQ ID NO: 3) of the modifiedenv gene m4070A;

FIG. 17 which shows a restricted gene expression construct; 4070AEnvelope to a first cell; p450 to a second cell;

FIG. 18 which shows the use of an intron to restrict NOI (in thisexample p450) expression to a transduced cell;

FIG. 19 which shows a pictorial representation of the Transfervector-pE1sp1A;

FIG. 20 which shows a pictorial representation of pE1sp1A construct;

FIG. 21 which shows a pictorial representation of pE1RevE construct;

FIG. 22 which shows a pictorial representation of pE1HORSE3.1-gagpolconstruct;

FIG. 23 which shows a pictorial representation of pE1PEGASUS4-Genomeconstruct;

FIG. 24 which shows a pictorial representation of pCI-Neo construct;

FIG. 25 which shows a pictorial representation of pCI-Rab construct;

FIG. 26 which shows a pictorial representation of pE1Rab construct;

FIG. 27a is a schematic representation of the natural splicingconfiguration in a retroviral vector;

FIG. 27b is a schematic representation of the splicing configuration inknown retroviral vectors;

FIG. 27c is a schematic representation of the splicing configurationaccording to the present invention; and

FIG. 28 is a schematic representation of the dual hybrid viral vectorsystem according to the present invention.

In slightly more detail:

FIG. 1 shows the structure of a retroviral proviral genome. In thisregard, the simplest retroviruses such as the murine oncoretroviruseshave three open reading frames; gag, pol and env. Frameshift during gagtranslation leads to pol translation. Env expression and translation isachieved by splicing between the splice donor (SD) and splice acceptor(SA) shown. The packaging signal is indicated as Psi and is onlycontained in the full length transcripts—not the env expressingsub-genomic transcripts where this signal is removed during the splicingevent.

FIG. 2 schematically shows the addition of small T splice donor to pLTR.Here, the small-t splice donor sequence is inserted into an LTR vectordownstream of the start of transcription but upstream of R sequence suchthat upon reverse transcription (in the final construct) the U3-splicedonor-R cassette is ‘inherited’ to 5′ end of the proviral vector and RNAtranscripts expressed contain a splice donor sequence near their 5′terminus.

FIG. 3 shows a schematic diagram of pL-SA-N. Both the consensus spliceacceptor (T/C)nNC/TAG-G (Mount 1982 Nucleic Acids Res 10: 459-472) andbranch point are shown in underline and bold The arrow indicates theintron/exon junction. Here, the consensus splice acceptor sequence isinserted into the Stu1/BamH1 sites of pLXSN. By such positioning thisacceptor will therefore interact with any upstream splice donor (in thefinal RNA transcripts).

FIG. 4 shows a schematic diagram for the construction of pL-SA-N with asplice donor deletion. The gT to gC change is made by performing a PCRreaction on the pL-SA-N vector with the two oligonucleotides shownbelow. The resulting product is then cloned Spe1-Asc1 into pL-SA-N thusreplacing the wild-type splice donor gT with gC. Both Spe1 and Asc1sites are shown in bold and the mutation in the Spe1 oligonucleotideshown in captial bold.

FIG. 5 shows the sequence of MLV pICUT.

FIG. 6 shows a schematic diagram of the insertion of splice donor atCMV/R junction of EIAV LTR plasmid. PCR is performed with the twooligonulceotides outlined below and the resulting PCR product clonedSac1-BamH1 into CMVLTR with the equivalent piece removed. In the Sac1oligonucleotide the arrow indicates the start of transcription, the newinsert is shown in capital with splice donor sequence underlined. Thestart of R is shown in italics.

FIG. 7 shows a schematic diagram of the insertion of splice acceptorinto pEGASUS-1. Here, the double stranded oligonucleotide describedbelow is inserted into Xho1-Bpu1102 digested pEGASUS-i to generateplasmid PEGASUS+SA. Both consensus splice acceptor (T/C)nNC/TAG-G (Mount1982 ibid) and branch point are shown in underline and bold. The arrowindicates the intron/exon junction.

FIG. 8 shows a schematic diagram of the removal of wild-type splicedonor from EIAV vector. Splice donor sequence removed by overlapping PCRusing the oliognucleotides described below and the template pEGASUS+SA.First separate PCR reactions are performed with oligos1+2 and oligos3+4.The resulting amplified products are then eluted and used combined in athird PCR reaction. After 10 cycles of this third reaction oligo2 and 4are then added. The resulting product is then cloned Sac1-Sal1 intopEGASUS+SA to create the plasmid pEGASUS+SA(noSD). The position of thesplice donor (SD) is indicated. The point mutation changing thewild-type splice donor from GT to GC is shown in bold both in oligol andthe complementary oligo3.

FIG. 9 shows a schematic diagram of combining pCMVLTR+SD withpEGASUS+SA(noSD) to create pEICUT-1. Here, one inserts the Mil1 fragmentof pEGASUS+SA(noSD) into the unique Mil1 site of pCMV-LTR.

FIG. 10 shows a schematic diagram of the construction of pEICUT-LacZ. Itis made by the insertion of the Xho1-Bpu1102 LacZ fragment frompEGASUS-1 and inserting it into the Xho1-Bpu1102 site of pEICUT-1 asoutlined below.

FIG. 11 shows the pEICUT-LacZ sequence.

FIG. 12 shows a schematic diagram of the vector configuration in bothtransfected and transduced cells. Here, the starting pICUT vectorcontains no splice donor upstream of a splice acceptor (in this instancethe consensus splice acceptor derived from IgSA) and therefore theresulting RNA transcripts will not be spliced. Thus all transcripts willbe full length transcripts containing a packaging signal (A). Upontransduction however the splice donor (in this instance the small-Tspliced donor) is ‘inherited’ to the 5′ of the proviral vector such thatall RNA transcripts now produced contain splice donor upstream of asplice acceptor i.e. an intron and thus maximal splicing achieved (B).

FIG. 13 shows a schematic diagram of the restriction of gene expressionto either packaging and transduced cells. Restriction of gene expressionin this instance is achieved by placing the hygromycin ORF upstream ofthe neomycin ORF in MLV pEICUT (a). By this cloning strategy theresulting vector will now express RNA transcripts that expresshygromycin only in transfected cells because ribosome 5′ cap-dependenttranslation will read only the upstream ORF efficiently. However upontransduction hygromycin is now contained within a functional intron andis thus deleted from mature transcripts (b) and thus neomycin ORF is nowtranslated in a 5′ cap-dependent manner.

FIG. 14 shows a schematic diagram of the construction of a MLV pICUTNeo-p450 vector that restricts hygromycin expression to producer cellsand 2B6 (a p450 isoform) expression to transduced cells. The startingvector for this construction is the pICUT vector of FIG. 13 containingboth hygro and neo. The neo gene is replaced with the complete p450 2B6cDNA as follows: The complete 2B6 cDNA is obtained by RT-PCR on humanliver RNA (Clontech) using the following primers (SEQ ID NOS 21 and 22,respectively, in order of appearance);

5′ttcgatgatcaccaccatggaactcagcgtcctcctcttccttgcac3′

5′ttcgagccggctcatcagcggggcaggaagcggatctggtatgttg3′

This generates the complete 2B6 cDNA with an optimised kosak sequenceflanked with unique Bcl1 and NgoM1 sites. This cDNA is then cloned intothe Bcl1-NgoM1 site of pICUT-Hyg-Neo thus replacing Neo with p450 (see(A) below). Also shown below are the proviral DNA constructs in bothtransfected (B) and transduced (C) cells.

FIG. 15 is a sequence comparison of mutant env (m4070A) with wild typeMMLV sequence from the 3′ end of the pol gene.

FIG. 16 is the complete sequence of altered 4070A.

FIG. 17 shows a gene restricted expression retroviral vector whereby thefirst NOI (the 4070A envelope ORF) is expressed in the initial vectorand the second NOI (in this instance p450) is expressed only aftervector replication. After replication the 4070A gene is located within afunctional intron and thus removed during RNA splicing.

FIG. 18 shows a retroviral expression vector whereby the 5′ end of thep450 gene (flush to a splice donor) is only found upstream of the 3′ endof the p450 gene (flush to SA) after replication and thus only afterreplication is a functional p450 gene expressed (from spliced mRNA).

EXAMPLES Example 1 Construction of a Split-intron MLV Vector

(i) Addition of Small-T Splice Donor:

The starting plasmid for this construct is pLXSN (Miller et al 1989ibid); Firstly this construct is digested with Nhe1 and the backbonere-ligated to create an LTR (U3-R-U5) plasmid. Into this plasmid is theninserted an oligonucleotide containing the splice donor sequence betweenthe Kpn1-Bbe1 sites. Also contained within this oligonucleotide,downstream of the splice donor is the MLV R sequence up to the Kpn1. Theresulting plasmid is named 3′LTR-SD (see FIG. 2).

(ii) Addition of Splice Acceptor:

The splice acceptor sequence used in this construct (including thebranch point- an A residue between 20 and 40 bases upstream of thesplice acceptor involved in intron lariat formation (Aebi et al 1987Trends in Genetics 3: 102-107) is derived from an immunoglobulin heavychain variable region mRNA (Bothwell et al 1981 Cell 24: 625-637) butwith a consensus/optimised acceptor site. Such a sequence signal is alsopresent in pCI (Promega). This acceptor sequence is firstly insertedinto the BamH1-Stu1 sites of pLXSN as double stranded oligonucleotide tocreate the vector pL-SA-N (note: SV40 promoter is lost from pLNSX duringcloning). See FIG. 3 for an outline of the cloning strategy.

(iii) Removal of Original Splice Donor from pL-SA-N.

The removal of the splice donor contained within the gag sequence ofpL-SA-N is achieved by PCR based site directed mutagenesis. Twooligonucleotides are used to PCR amplify the region spanning the Asc1and Spe1 uniques sites of pL-SA-N. Also incorporated in theSpe1-spanning olgonucleotide is the agGTaag to agGCaag change also foundin the splicing negative pBABE vectors (Morgenstern et al 1990 ibid).See FIG. 4 for cloning strategy outline.

(iv) Combining pL(noSD)-SA-N with 3′LTR-SD.

The pL(noSD)SA-N plasmid contains a normal MLV derived 3′LTR. This isreplaced with the 3′LTR-SD sequence by taking the Nhe1 insert frompL(noSD)SA-N and dropping it into the Nhel digested 3′LTR-SD vector. Theresulting plasmid, named pICUT (Intron Created Upon Transduction)contains all the features of this new generation of retroviral vector(see FIG. 5 for sequence data)

Example 2 Construction of a Split-intron Lentivector

Construction of Initial EIAV Lentiviral Expression Vector (also seepatent application GB9727135.7)

For the construction of a split-function lentiviral vector the startingpoint is the vector named pEGASUS-1 (see patent application GB9727135.7). This vector is derived from infectious proviral EIAV clonepSPEIAV19 (accession number: U01866; Payne et al 1994). Its constructionis outlined as follows: First; the EIAV LTR, amplified by PCR, is clonedinto pBluescript II KS+ (Stratagene). The MluI/MluI (216/8124) fragmentof pSEIAV19 is then inserted to generate a wild-type proviral clone(pONY2) in pBluescript II KS+ (FIG. 1). The env region is then deletedby removal of the Hind III/Hind III fragment to generate pONY2-H. Inaddition, a BglII/NcoI fragment within pol (1901/4949) is deleted and aβ-galactosidase gene driven by the HCMV IE enhancer/promoter inserted inits place. This is designated pONY2.10nlsLacZ. To reduce EIAV sequenceto 759 base pairs and to drive primary transcript off a CMV promoter:First; sequence encompassing the EIAV polypurine tract (PPT) and the3′LTR are PCR amplified from pONY2.10LacZ using primers (SEQ ID NOS 18and 19 respectively, in order of appearance):

PPTEIAV+ (Y8198): GACTACGACTAGTGTATGTTTAGAAAAACAAGG, and

3′NEGSpeI(Y8199): CTAGGCTACTAGTACTGTAGGATCTCGAACAG.

The PCR product is then cloned into the Spe1 site of pBS II KS⁺;orientated such that U5 is proximal to Not1 in the pBlueScript II KS⁺

Next, for the reporter gene cassette, a CMV promoter/LacZ from pONY2.10nlsLacZ is removed by Pst1 digest and cloned into the Pst1 site ofpBS.3′LTR orientated such that LacZ gene is proximal to the 3′LTR, thisvector is named pBS CMVLacZ.3′LTR.

The 5′region of the EIAV vector is constructed in the expression vectorpCIEneo which is derivative of pCIneo (Promega)-modified by theinclusion of approximately 400 base pairs derived from the 5′end of thefull CMV promoter as defined previously. This 400 base pair fragment isobtained by PCR amplifcation using primers (SEQ ID NOS 20 and 6respectively, in order of appearance):

VSAT1: (GGGCTATATGAGATGAATAATAAAATGTGT) and

VSAT2: (TATTAATAACTAGT) and

pHIT60 (Soneoka et al 1995 Nucleic Acids Res 23: 628-633) as template.The product is digested with BglII and SpeI and cloned into theBglII/SpeI sites of pCIE-Neo.

A fragment of the EIAV genome running from the R region to nt 150 of thegag coding region (nt 268 to 675) is amplified from pSEIAV with primers:

CMV5′EIAV2 (SEQ ID NO: 7):

(ZO591)(GCTACGCAGAGCTCGTTTAGTGAACCGGGCACTCAGATTCTG: (sequencesunderlined anneals to the EIAV R region) and (SEQ ID NO: 8)

3′PSI. NEG (GCTGAGCTCTAGAGTCCTTTTCTTTTACAAAGTTGG).

The resulting PCR product is flanked by Xba1 and Sac1 sites. This isthen cut and cloned into the pCIE-Neo Xba1-SacI sites. The resultingplasmid, termed pCIEneo5′EIAV now contains the start of the EIAV Rregion at the transcriptional start point of the CMV promoter. TheCMVLacZ/3LTR cassette is then inserted into the pCIEneo5′EIAV plasmid bytaking the Apa1 to Nor1 fragment from pBS.CMVLacZ.3LTR and cloning itinto the Sal1-Not1 digested pCIEneo.5′EIAV (the Sal1 and Apa1 sites isT4 “polished” to create blunt the ends prior to the vector and insertrespective Not1 digests). The resulting plasmid is named pEGASUS-1.

For use as a gene delivery vector pEGASUS-1 requires both gag/pol andenv expression provided in trans by a packaging cell. For the source ofgag/pol an EIAV gagpol expression plasmid (pONY3) is made by insertingthe Mlu I/Mlu I fragment from pONY2-H into the mammalian expressionplasmid pCI-neo (Promega) such that the gag-pol gene is expressed fromthe hCMV-MIE promoter-enhancer and contains no LTR sequences. For thesource of env; the pRV583 VSV-G expression plasmid is routinely used.These three vectors are used in a three plasmid co-transfection asdescribed for MLV-based vectors (Soneoka et al 1995 Nucl. Acids Res.23:628-633) the resulting virus routinely titres at between 10⁴ and 10⁵lacZ forming units per ml on D17 fibroblasts.

Construction of a EIAV Lentiviral Version Vector of pICUT; Named pEICUT

To construct pEICUT firstly pEGASUS-1 the Xma1-SexA1 fragment is removedfrom pEGASUS-1 and the ends ‘blunted’ with T4 polymerase and plasmidre-ligated to create a plasmid containg only the CMV-R-U5 part ofpEGASUS-1 which retains the SV40-Neo cassette in the backbone. Thisplasmid is named CMVLTR. To insert a splice donor at the CMV-R borderPCR is carried out with the two oligonucleotides shown below in FIG. 6and as outlined in the FIG. 6 legend. The resulting plasmid is namedpCMVLTR+SD. The same immunoglobulin based consensus splice acceptor asfor MLV pICUT (see earlier) is used in the EIAV version. This isinserted using oligonucleotides described in FIG. 7 into theXhoI-Bpu1102 site of pEGASUS-1 to create the plasmid pEGASUS+SA. Thewild-type splice donor of EIAV is removed by carrying out overlappingPCR with the oligonulceotides and methodology as described in FIG. 8,using pEGASUS+SA as a template to generate the plasmid pEGASUS+SA(noSD).To then create pEICUT-1, the Mlu1-Mlu1 fragment from pEGASUS+SA(noSD) isthen inserted into the unique Mlu1 site of pCMVLTR+SD to generatepEICUT-1 (see FIG. 9). LacZ can be then transferred from pEGASUS-1 intopEICUT-1 by Xho1-Bpu1102 digest and insertion to create pEICUT-Z (seeFIG. 10; for sequence data see FIG. 11).

Both the MLV and EIAV pICUT vectors contain a strong splice acceptorupstream of the splice donor and therefore no functional intron (intronsrequire splice donors positioned 5′ of splice acceptors). For thisreason, when the vector is transfected into producer cells the resultingtranscripts generated will not be spliced. Thus the packaging signalwill not be lost and as a consequence maximal packaging is achievable(see FIG. 12).

However because of the unique way by which retroviruses replicate, upontransduction, transcripts generated from the integrated pICUT vectorwill differ from those of transfected cells described above. This isbecause during replication the 3′U3 promoter (up to the 5′start of R) iscopied and used as the 5′ promoter in transduced cells. For this reasontranscripts generated from integrated pICUT will now contain a strongsplice donor 5′ of a strong splice acceptor, both of which being locatedupstream of the neo ORF. Such transcripts will therefore contain afunctional intron in the 5′UTR (untranslated region) and thus bemaximally spliced and translated.

Another advantage of such vectors described above is that because theintron is created only upon transduction it is possible to limit geneexpression to either packaged or transduced cells. One example of howthis is achieved is outlined in FIGS. 13. The strategy entails thecloning of a second gene (in this example hygromycin) upstream of thesplice acceptor. This is achieved by taking out the hygromycin cDNA on aSalI fragment from SelctaVector Hygro (Ingenius; Oxfordshire, UK), andcloning this into a Xho1 site (located upstream of the splice acceptor)of pICUT. This vector selectively expresses hygromycin in thetransfected cells and neomycin in transduced cells. The reason for thisis that in any one mRNA transcript only the first gene is translated bythe ribsome without the aid of internal ribosome binding sites (IRESs).In the transfected cell this gene will be hygromycin. However in thetransduced cells because the hygromycin open reading frame (ORF) iscontained within a functional intron this gene will now be removed frommature mRNA transcripts thus allowing neo ORF translation.

Vectors with such cell specific gene expression maybe of clinical usefor a variety of reasons; By way of example, expression of resistancemarkers can be restricted to producer cells-where they are required andnot in transduced cells where they may be immunogenic. By way of anotherexample, expression of toxic genes such as ricin and dominant negativesignalling proteins could be restricted to transduced cells where theymay be required to optionally arrest cell growth or kill cells but notin producer cells-where such features would prevent high titre virusproduction. FIG. 14 shows a Neo-p450 MLV pICUT construct such that onlyNeo is expressed in producer cells and the pro-drug p450 2B6 isoformexpressed in transduced cells.

Another benefit of creating an intron upon transduction is that anyessential elements required for vector function can now be placed insidea functional intron, which is created upon transduction, and be removedfrom transduced cell transcripts. By way of example, with both the MLVand the lentivector pICUT vectors, the viral transcript contained thefunctional Psi packaging signal (see Bender et al 1987 for the positionof Psi in MLV; see patent application GB 9727135.7 for position of Psiin EIAV) within an intron which was created upon transduction andremoved from the transduced cell transcripts.

The benefits from such an arrangement include:

(i) Enhanced translation from resulting transcripts because ribosomesmay “stutter” in the presence of a Psi secondary structure- if present(Krall et al 1996 ibid and reference therein).

(ii) In the absence of the packaging signal, transcript packaging byendogenous retroviruses is prevented.

(iii) Unwanted premature translation initiation is prevented when viralessential elements such as gag (and other potential ATG translationstart sites) are removed from the transcripts expressed in transducedcells. This is of particular benefit when packaging signals extend intogag as is the case for both the EIAV and MLV pICUT vectors.

(iv) Promoter, enhancers and suppressors may be placed within an introncreated upon transduction thus mimicking other transcript arrangementslike those generated from CMV that contain such entities within introns(Chapman et al 1991 ibid)

In summation the novel pICUT vector system described in the presentinvention facilitates the following arrangments:

(I) Maximal packaging and reduced translation of transcripts in producercells.

(ii) Maximal splicing and therefore intron enhanced translation oftranscripts in transduced cells

(iii) Restriction of gene and/or viral essential element expression toeither producer or transduced cells.

Example 3 Construction of an MMLV Amphotropic env Gene with MinimalHomology to the pol Gene and a gag-pol Transcription Cassette

In the Moloney murine leukaemia virus (MMLV), the first approximately 60bps of the env coding sequence overlap with sequences at the 3′ end ofthe pol gene. The region of homology between these two genes was removedto prevent the possibility of recombination between them in cellsexpressing both genes.

The DNA sequence of the first 60 bps of the coding sequence of env waschanged while retaining the amino acid sequence of the encoded proteinas follows. A synthetic oligonucleotide was constructed to alter thecodon usage of the 5′-end of env (See FIG. 15) and inserted into theremainder of env as follows.

The starting plasmid for re-construction of the 5′ end of the 4070A genewas the pCI plasmid (Promega) into which had previously been cloned thexba1-Xba1 fragment containing the 4070A gene from pHIT456 (Soneoka et al1995 ibid) to form pCI-4070A.

A PCR reaction was performed with primers A and B (FIG. 15) on pCI4070Ato produce a 600 base pair product. This product was then cloned betweenthe Nhe1 and Xho1 sites of pCI4070A. The resulting construct wassequenced across the Nhe1/Xho1 region. Although the amino acid sequenceof the resulting gene is the same as the original 4070A, the region ofhomology with the pol gene is removed.

The complete sequence of the modified env gene m4070A is given in FIG.16. This sequence is inserted into the expression vector pCI (Promega)by standard techniques.

The CMV gag-pol transcription unit is obtaind from pHIT60 (Soneoka et al1995 ibid).

Example 4 Deletion of gag Sequences from the Retroviral Packaging Signal

A DNA fragment containing the LTR and minimal functional packagingsignal is obtained from the retroviral vector MFG (Bandara et al 1993Proc Natl Acad Sci 90: 10764-10768) or MMLV proviral DNA by PCR reactionusing the following oligonucleotide primers:

HindIIIR(SEQ ID NO: 9) GCATTAAAGCTTTGCTCT

L523(SEQ ID NO: 10) GCCTCGAGCAAAAATTCAGACGGA

This PCR fragment contains MMLV nucleotides +1 to +523 and thus does notcontain gag coding sequences which start at +621 (numbering based on thenucleotide sequence of MMLV Shinnick et al 1981 Nature 293: 543-548).

The PCR fragment can be used to construct a retroviral genome vector bydigestion using HindIII and Xho1 restriction enzymes and sub-cloningusing standard techniques. Such vectors contain no homology with gagcoding sequences.

Example 5 Construction of Defective Retroviral Genome

The transcription unit capable of producing a defective retroviralgenome is shown in FIG. 17. It contains the following elements: ahypoxia regulated promoter enhancer comprising 3 copies of the PGK—geneHRE and a SV40 promoter deleted of the 72bp-repeat enhancer from pGL3(Promega); a MMLV sequence containing R, U5 and the packaging signal;the coding sequence of m4070A (Example 3); a splice acceptor; a cloningsite for insertion of a coding sequence for a therapeutic protein; thepolypyrimidine tract from MMLV; a second copy of the HRE-containingpromoter-enhancer; a splice donor site; and a second copy of R, U5.

On reverse transcription and integration of the vector into thesecondary target cell, the splice donor is introduced upstream of theenv gene causing it to be removed from mRNA by splicing and therebypermitting efficient expression of the therapeutic gene only in thesecondary target cell (See FIG. 17).

Example 6 Construction of a Conditional Expression Vector for CytochromeP450

FIG. 18 shows the structure of retroviral expression vector cDNA codingsequences from the cytochrome P450 gene in two halves such that onlyupon transduction is the correct splicing achieved to allow P450expression. This therefore restricts expression to transduced cells.

1) The starting plasmid for the construction of this vector is pLNSX(Miller and Rosman 1989 BioTechniques 7: 980-990). The natural splicedonor ( . . . agGTaag . . . ) contained within the packaging signal ofpLNSX (position 781/782) is mutated by PCR mutagenesis using the ALTEREDSITES II mutageneisis kit (Promega) and a synthetic oligonucleotide ofthe sequencer(SEQ ID NO: 11):

5′-caaccaccgggagGCaagctggccagcaacuta-3′

2) A CMV promoter from the pCI expression vector (Promega) is isolatedby PCR using the following two oligonucleotides:

Primer 1(SEQ ID NO: 12): 5′-atcggctagcagatcttcaatattggccattagccatat-3′

Primer 2(SEQ ID NO: 13):5′-atcgagatctgcggccgcttacctgcccagtgcctcacgaccaa-3′

This produces a fragment containing the CMV promoter with a 5′Nhe1 site(Primer 1) and a 3′Not1 and Xba1 site (Primer 2). It is cut with Nhe1and Xba1 and cloned into pLNSX from which an Nhe1-Nhe1 fragment has beenremoved.

3) The 5′ end of a cytochrome P450 cDNA coding sequence is isolated byRT-PCR from human liver RNA (Clontech) with the following primers:

Primer 3(SEQ ID NO: 14):5′-atcggcggccgcccaccatggaactcagcgtcctcctcttccttgcaccctagg-3′

Primer 4(SEQ ID NO: 15):5′-atcggcggccgcacttacCtgtgtgccccaggaaagtatttcaagaagccag-3′

This amplifies the 5′ end of the p450 from the ATG to residue 693(numbering from the translation initiation site Yamano et al 1989Biochem 28:7340-7348). Contained on the 5′ end of the fragment (derivedfrom Primer 3) is also a Not1 site and an optimised “Kozak” translationinitiation signal. Contained on the 3′ end of the sequence (derived fromprimer 4) is another Not1 site and a consensus splice donor sequence(also found in pCI and originally derived from the human beta globingene) with the GT splice donor pair located flush against residue 704 ofP450 (the complementary residue is shown in uppercase in Primer 4). Thisfragment is digested with Not1 and cloned into the Not1 digested plasmidgenerated in step 2.

The Nhe1-Nhe1 fragment removed during the cloning of step 2 is thenre-introduced into the plasmid of step 3. This creates a retroviralvector as described in FIG. 17 but missing the 3′ end P450.

The 3′of the P450 coding sequence is isolated by RT-PCR amplificationfrom human liver RNA (Clontech) using the following primers:

Primer 5(SEQ ID NO: 16):actgtgatcataggcacctattggtcttactgacatccactttctctccacagGcaagtttacaaaacctgcaggaaatcaatgcttacatt-3′

Primer 6(SEQ ID NO: 17): actgatcgamccctcagccccttcagcggggcaggaagc-3′

This generates the PCR amplified 3′ end of P450 from residue 705 (inuppercase primer 5) and extends past the translation termination codon.Contained within the 5′ end of this product and generated by primer 5 isa Bcl1 restriction site and a consensus splice acceptor and branch point(also found in pCI and originally from an immunoglobulin gene) upstreamof residue 705. Contained at the 3′ end of this product downstream ofthe stop codon and generated by primer 6 is a Cla1 site. This PCRproduct is then digested with Bcl1 and Cla1 and cloned into the vectorof step 3 with the Bcl1-Cla1 fragment removed to generate the retroviralvector as shown in FIG. 18.

The following examples describe the construction of an adenolentiviralsystem that can be used for the transient production of lentivirus invitro or in vivo.

First Generation Recombinant Adenovirus

The first generation adenovirus vectors consist of a deletion of the E1and E3 regions of the virus allowing insertion of foreign DNA, usuallyinto the left arm of the virus adjacent to the left Inverted TerminalRepeat (ITR). The viral packaging signal (194-358 nt) overlaps with theE1 a enhancer and hence is present in most E1 deleted vectors. Thissequence can be translocated to the right end of the viral genome(Hearing & Shenk,1983 Cell 33: 59-74). Therefore, in an E1 deletedvector 3.2 kb can be deleted (358-3525 nt).

Adenovirus is able to package 105% length of the genome, thus allowingfor addition of an extra 2.1 kb. Therefore, in an E1/E3 deleted viralvector the cloning capacity becomes 7-8 kb (2.1 kb+1.9 kb (removal ofE3)+3.2 kb (removal of E1). Since the recombinant adenovirus lacks theessential E1 early gene it is unable to replicate in non-E1complementing cell lines. The 293 cell line was developed by Graham etal. (1977 J Gen Virol 36: 59-74) and contains approximately 4 kb fromthe left end of the Ad5 genome including the ITR, packaging signal, E1a,E1b and pIX. The cells stably express E1a and E1b gene products, but notthe late protein IX, even though pIX sequences are within E1b. Innon-complementing cells the E1 deleted virus transduces the cell and istransported to the nucleus but there is no expression from the E1deleted genome.

First Generation Adenovirus Production System

Microbix Biosystems—nbl Gene Sciences

The diagram in FIG. 19 shows the general strategy used to createrecombinant adenoviruses using the microbix system

The general strategy involves cloning the foreign DNA into an E1 shuttlevector, where the E1 region from 402-3328 bp is replaced by the foreignDNA cassette. The recombinant plasmid is then co-transfected into 293cells with the pJM17 plasmid. pJM17 contains a deletion of the E3 regionand an insertion of the prokaryotic pBRX vector (including theampicillin resistance and bacterial ori sequences) into the E1 region at3.7 map units. This 40 kb plasmid is therefore too large to be packagedinto adeno nucleocapsids but can be propagated in bacteria Intracellularrecombination in 293 cells results in replacement of the amp^(r) and orisequences with the insert of foreign DNA.

Example 7 Construction of Transfer Plasmids for the Creation ofAdenoviruses Containing EIAV Components

In order to produce lentiviral vectors four adenovirus need to be made:genome, gagpol, envelope (rabies G) and Rev. The lentiviral componentsare expressed from heterologous promoters they contain introns whereneeded (for high expression of gagpol, Rev and Rabies envelope) and apolyadenylation signal. When these four viruses are transduced into E1aminus cells the adenoviral components will not be expressed but theheterlogous promoters will allow the expression of the lentiviralcomponents. An example is outlined below (example 1) of the constructionof an EIAV adenoviral system (Application number: 9727135.7). The EIAVis based on a minimal system that is one lacking any of thenon-essential EIAV encoded proteins (S2, Tat or envelope). The envelopeused to pseudotype the EIAV is the rabies envelope (G protein). This hasbeen shown to pseudotype EIAV well (Application number. 9811152.9).

Transfer Plasmids

Decribed below is the construction of the transfer plasmids containingthe EIAV components. The transfer plamsid is pE1sp1A (FIG. 20).

The recombinant transfer plamsids can the be used to make recombinantadenoviruses by homologous recombination in 293 cells.

A pictorial representation of the following plasmids is attached.

A) pE1RevE—Rev Construct

The plasmid pCI-Rev is cut with Apa LI and Cla I. The 2.3 kb bandencoding EIAV Rev is blunt ended with Klenow polymerase and insertedinto the Eco RV site of pE1 sp1A to give plasmid pE1RevE (FIG. 21).

B) pE1HORSE3.1—gagpol Construct

pHORSE3.1 was cut with Sna BI and Not I. The 6.1 kb band encoding EIAVgagol was inserted into pE1RevE cut with Sna BI and Not I (7.5 kb bandwas purified). This gives plasmid pE1HORSE3.1 (FIG. 22).

C) pE1PEGASUS—Genome Construct

pEGASUS4 was cut with Bgl II and Not I. The 6.8 kb band containing theEIAV vector genome was inserted into pE1RevE cut with Bgl II and Not I(6.7 kb band was purified). This gave plasmid pE1PEGASUS (FIG. 23).

D) pCI-Rab—Rabies Construct

In order to make pE1Rab the rabies open reading frame was inserted intopCI-Neo (FIG. 24) by cutting plasmid pSA91RbG with Nsi I and Ahd I. The1.25 kb band was bluntended with T4 DNA polymerase and inserted intopCI-Neo cut with Sma I. This gave plasmid pCI-Rab (FIG. 25).

F) pE1Rab—Rabies construct

pCI-Rab was cut with Sna BI and Not I. The 1.9 b band encoding Rabiesenvelope was inserted into pE1RevE cut with Sna BI and Not I (7.5 b bandwas purified). This gave plasmid pE1Rab (FIG. 26).

SUMMARY

The present invention relates to a novel delivery system suitable forintroducing one or more NOIs into a target cell.

In one preferred aspect the present invention covers a retroviral vectorcomprising a functional splice donor site and a functional spliceacceptor site; wherein the functional splice donor site and thefunctional splice acceptor site flank a first nucleotide sequence ofinterest (“NOI”); wherein the functional splice donor site is upstreamof the functional splice acceptor site; wherein the retroviral vector isderived from a retroviral pro-vector; wherein the retroviral pro-vectorcomprises a first nucleotide sequence (“NS”) capable of yielding thefunctional splice donor site and a second NS capable of yielding thefunctional splice acceptor site; wherein the first NS is downstream ofthe second NS; such that the retroviral vector is formed as a result ofreverse transcription of the retroviral pro-vector.

Alternatively expressed, this aspect covers a novel delivery systemwhich comprises one or more NOIs flanked by a functional SD and SAprovided that this has been generated from a pro-vector in which theorder of the SD and SA is reversed to render the splicingnon-functional.

This aspect of the present invention can be called the“split-intron”*aspect. A schematic diagram showing this aspect of thepresent invention is provided in FIG. 27c. In contrast, FIGS. 27a and 27b show splicing configurations in known retroviral vectors.

Another broad aspects of the present invention include a novel deliverysystem which comprises one or more adenoviral vector components capableof packaging one or more lentiviral vector components, whereinoptionally the lentiviral vector comprises a split intron configuration.

This aspect of the present invention in the general sense can be calleda hybrid viral vector system. In this particular case, the combinationof an adenoviral component and a lentiviral component can be called adual hybrid viral vector system.

A schematic diagram showing this aspect of the present invention isprovided in FIG. 28.

These and other broad aspects of the present invention are discussedherein.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

36 1 5689 DNA Artificial Sequence Description of Artificial Sequence MLVpICUT 1 gctagcttaa gtaacgccac tttgcaaggc atggaaaaat acataactgagaatagaaaa 60 gttcagatca aggtcaggaa caaagaaaca gctgaatacc aaacaggatatctgtggtaa 120 gcggttcctg ccccggctca gggccaagaa cagatgagac agctgagtgatgggccaaac 180 aggatatctg tggtaagcag ttcctgcccc ggctcggggc caagaacagatggtccccag 240 atgcggtcca gccctcagca gtttctagtg aatcatcaga tgtttccagggtgccccaag 300 gacctgaaaa tgaccctgta ccttatttga actaaccaat cagttcgcttctcgcttctg 360 ttcgcgcgct tccgctctcc gagctcaata aaagagccca caacccctcactcggcgcgc 420 cagtcttccg atagactgcg tcgcccgggt acccgtattc ccaataaagcctcttgctgt 480 ttgcatccga atcgtggtct cgctgttcct tgggagggtc tcctctgagtgattgactac 540 ccacgacggg ggtctttcat ttgggggctc gtccgggatt tggagacccctgcccaggga 600 ccaccgaccc accaccggga ggcaagctgg ccagcaactt atctgtgtctgtccgattgt 660 ctagtgtcta tgtttgatgt tatgcgcctg cgtctgtact agttagctaactagctctgt 720 atctggcgga cccgtggtgg aactgacgag ttctgaacac ccggccgcaaccctgggaga 780 cgtcccaggg actttggggg ccgtttttgt ggcccgacct gaggaagggagtcgatgtgg 840 aatccgaccc cgtcaggata tgtggttctg gtaggagacg agaacctaaaacagttcccg 900 cctccgtctg aatttttgct ttcggtttgg aaccgaagcc gcgcgtcttgtctgctgcag 960 cgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtatttgtctgaaaat 1020 tagggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaaagatgtcgag 1080 cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttaccttctgctct 1140 gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaaccgagacctc 1200 atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacacccagaccaggtc 1260 ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggtcaagcccttt 1320 gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctcccccttgaacct 1380 cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttctctaggcgcc 1440 ggaattcgtt aactcgagga tctaacctag gtctcgagtg tttaaacactgggcttgtcg 1500 agacagagaa gactcttgcg tttctgatag gcacctattg gtcttactgacatccacttt 1560 gcctttctct ccacaggtga ggcctaggct tttgcaaaaa gcttgggctgcaggtcgagg 1620 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaagatggattgca 1680 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactgggcacaacagac 1740 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcccggttctttt 1800 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcagcgcggctatc 1860 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtcactgaagcggg 1920 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcatctcaccttgc 1980 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcatacgcttgatcc 2040 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcacgtactcggat 2100 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggctcgcgccagc 2160 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcgtcgtgaccca 2220 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctggattcatcga 2280 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggctacccgtgatat 2340 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacggtatcgccgc 2400 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttctgagcgggact 2460 ctggggttcg ataaaataaa agattttatt tagtctccag aaaaaggggggaatgaaaga 2520 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaaggcatggaaaaa 2580 tacataactg agaatagaga agttcagatc aaggtcagga acagatggaacagctgaata 2640 tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggccaagaacaga 2700 tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcctgccccggctc 2760 agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttctagagaacca 2820 tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttatttgaactaac 2880 caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctcaataaaagag 2940 cccacaaccc ctcactcggg gcgccgttaa cactagtaag cttgctctaaggtaaatatg 3000 tcgacaggcc tgcgccagtc ctccgattga ctgagtcgcc cgggtacccgtgtatccaat 3060 aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggagggtctcctct 3120 gagtgattga ctacccgtca gcgggggtct ttcatttggg ggctcgtccgggatcgggag 3180 acccctgccc agggaccacc gacccaccac cgggaggtaa gctggctgcctcgcgcgttt 3240 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtcacagcttgtct 3300 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtgttggcgggtg 3360 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactggcttaactat 3420 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaataccgcacaga 3480 tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcactgactcgctg 3540 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggtaatacggtta 3600 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggccagcaaaaggcc 3660 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgcccccctgacgag 3720 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggactataaagatac 3780 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccctgccgcttacc 3840 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatagctcacgctgt 3900 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgcacgaacccccc 3960 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaacccggtaaga 4020 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagcgaggtatgta 4080 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactagaaggacagta 4140 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttggtagctcttga 4200 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagcagcagattacg 4260 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtctgacgctcag 4320 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaaggatcttcacc 4380 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatatatgagtaaact 4440 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgatctgtctattt 4500 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacgggagggctta 4560 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggctccagattta 4620 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgcaactttatcc 4680 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttcgccagttaat 4740 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctcgtcgtttggt 4800 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatcccccatgttg 4860 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaagttggccgca 4920 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcatgccatccgta 4980 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaatagtgtatgcgg 5040 cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccacatagcagaact 5100 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaaggatcttaccg 5160 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttcagcatctttt 5220 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgcaaaaaaggga 5280 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatattattgaagc 5340 atttatcagg gttattgtct catgagcgga tacatatttg aatgtatttagaaaaataaa 5400 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtctaagaaaccatt 5460 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcgtcttcaagaa 5520 ttcataccag atcaccgaaa actgtcctcc aaatgtgtcc ccctcacactcccaaattcg 5580 cgggcttctg cctcttagac cactctaccc tattccccac actcaccggagccaaagccg 5640 cggcccttcc gtttctttgc ttttgaaaga ccccacccgt aggtggcaa5689 2 9756 DNA Artificial Sequence Description of Artificial SequencepEICUT-LacZ 2 tgaataataa aatgtgtgtt tgtccgaaat acgcgttttg agatttctgtcgccgactaa 60 attcatgtcg cgcgatagtg gtgtttatcg ccgatagaga tggcgatattggaaaaattg 120 atatttgaaa atatggcata ttgaaaatgt cgccgatgtg agtttctgtgtaactgatat 180 cgccattttt ccaaaagtga tttttgggca tacgcgatat ctggcgatagcgcttatatc 240 gtttacgggg gatggcgata gacgactttg gtgacttggg cgattctgtgtgtcgcaaat 300 atcgcagttt cgatataggt gacagacgat atgaggctat atcgccgatagaggcgacat 360 caagctggca catggccaat gcatatcgat ctatacattg aatcaatattggccattagc 420 catattattc attggttata tagcataaat caatattggc tattggccattgcatacgtt 480 gtatccatat cgtaatatgt acatttatat tggctcatgt ccaacattaccgccatgttg 540 acattgatta ttgactagtt attaatagta atcaattacg gggtcattagttcatagccc 600 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggctgaccgcccaa 660 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgccaatagggac 720 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttggcagtacatca 780 agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaatggcccgcctg 840 gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtacatctacgtatt 900 agtcatcgct attaccatgg tgatgcggtt ttggcagtac accaatgggcgtggatagcg 960 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatgggagtttgttttg 1020 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tgcgatcgcccgccccgttg 1080 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagctcgtttagtg 1140 aaccgggcac tcagattctg cggtctgagt cccttctctg ctgggctgaaaaggcctttg 1200 taataaatat aattctctac tcagtccctg tctctagttt gtctgttcgagatcctacag 1260 ttggcgcccg aacagggacc tgagaggggc gcagacccta cctgttgaacctggctgatc 1320 gtaggatccc cgggacagca gaggagaact tacagaagtc ttctggaggtgttcctggcc 1380 agaacacagg aggacaggta agatgggaga ccctttgaca tggagcaaggcgctcaagaa 1440 gttagagaag gtgacggtac aagggtctca gaaattaact actggtaactgtaattgggc 1500 gctaagtcta gtagacttat ttcatgatac caactttgta aaagaaaaggactctagagt 1560 cgaccccctc gacgtttaaa cactgggctt gtcgagacag agaagactcttgcgtttctg 1620 ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacaggtcacgtgaa 1680 gctagcctcg aggatctgcg gatccgggga attccccagt ctcaggatccaccatggggg 1740 atcccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaacttaatcgcc 1800 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgcaccgatcgcc 1860 cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggtttccggcaccag 1920 aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatactgtcgtcgtcc 1980 cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgtaacctatccca 2040 ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttactcgctcacat 2100 ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattatttttgatggcgtta 2160 actcggcgtt tcatctgtgg tgcaacgggc gctgggtcgg ttacggccaggacagtcgtt 2220 tgccgtctga atttgacctg agcgcatttt tacgcgccgg agaaaaccgcctcgcggtga 2280 tggtgctgcg ttggagtgac ggcagttatc tggaagatca ggatatgtggcggatgagcg 2340 gcattttccg tgacgtctcg ttgctgcata aaccgactac acaaatcagcgatttccatg 2400 ttgccactcg ctttaatgat gatttcagcc gcgctgtact ggaggctgaagttcagatgt 2460 gcggcgagtt gcgtgactac ctacgggtaa cagtttcttt atggcagggtgaaacgcagg 2520 tcgccagcgg caccgcgcct ttcggcggtg aaattatcga tgagcgtggtggttatgccg 2580 atcgcgtcac actacgtctg aacgtcgaaa acccgaaact gtggagcgccgaaatcccga 2640 atctctatcg tgcggtggtt gaactgcaca ccgccgacgg cacgctgattgaagcagaag 2700 cctgcgatgt cggtttccgc gaggtgcgga ttgaaaatgg tctgctgctgctgaacggca 2760 agccgttgct gattcgaggc gttaaccgtc acgagcatca tcctctgcatggtcaggtca 2820 tggatgagca gacgatggtg caggatatcc tgctgatgaa gcagaacaactttaacgccg 2880 tgcgctgttc gcattatccg aaccatccgc tgtggtacac gctgtgcgaccgctacggcc 2940 tgtatgtggt ggatgaagcc aatattgaaa cccacggcat ggtgccaatgaatcgtctga 3000 ccgatgatcc gcgctggcta ccggcgatga gcgaacgcgt aacgcgaatggtgcagcgcg 3060 atcgtaatca cccgagtgtg atcatctggt cgctggggaa tgaatcaggccacggcgcta 3120 atcacgacgc gctgtatcgc tggatcaaat ctgtcgatcc ttcccgcccggtgcagtatg 3180 aaggcggcgg agccgacacc acggccaccg atattatttg cccgatgtacgcgcgcgtgg 3240 atgaagacca gcccttcccg gctgtgccga aatggtccat caaaaaatggctttcgctac 3300 ctggagagac gcgcccgctg atcctttgcg aatacgccca cgcgatgggtaacagtcttg 3360 gcggtttcgc taaatactgg caggcgtttc gtcagtatcc ccgtttacagggcggcttcg 3420 tctgggactg ggtggatcag tcgctgatta aatatgatga aaacggcaacccgtggtcgg 3480 cttacggcgg tgattttggc gatacgccga acgatcgcca gttctgtatgaacggtctgg 3540 tctttgccga ccgcacgccg catccagcgc tgacggaagc aaaacaccagcagcagtttt 3600 tccagttccg tttatccggg caaaccatcg aagtgaccag cgaatacctgttccgtcata 3660 gcgataacga gctcctgcac tggatggtgg cgctggatgg taagccgctggcaagcggtg 3720 aagtgcctct ggatgtcgct ccacaaggta aacagttgat tgaactgcctgaactaccgc 3780 agccggagag cgccgggcaa ctctggctca cagtacgcgt agtgcaaccgaacgcgaccg 3840 catggtcaga agccgggcac atcagcgcct ggcagcagtg gcgtctggcggaaaacctca 3900 gtgtgacgct ccccgccgcg tcccacgcca tcccgcatct gaccaccagcgaaatggatt 3960 tttgcatcga gctgggtaat aagcgttggc aatttaaccg ccagtcaggctttctttcac 4020 agatgtggat tggcgataaa aaacaactgc tgacgccgct gcgcgatcagttcacccgtg 4080 caccgctgga taacgacatt ggcgtaagtg aagcgacccg cattgaccctaacgcctggg 4140 tcgaacgctg gaaggcggcg ggccattacc aggccgaagc agcgttgttgcagtgcacgg 4200 cagatacact tgctgatgcg gtgctgatta cgaccgctca cgcgtggcagcatcagggga 4260 aaaccttatt tatcagccgg aaaacctacc ggattgatgg tagtggtcaaatggcgatta 4320 ccgttgatgt tgaagtggcg agcgatacac cgcatccggc gcggattggcctgaactgcc 4380 agctggcgca ggtagcagag cgggtaaact ggctcggatt agggccgcaagaaaactatc 4440 ccgaccgcct tactgccgcc tgttttgacc gctgggatct gccattgtcagacatgtata 4500 ccccgtacgt cttcccgagc gaaaacggtc tgcgctgcgg gacgcgcgaattgaattatg 4560 gcccacacca gtggcgcggc gacttccagt tcaacatcag ccgctacagtcaacagcaac 4620 tgatggaaac cagccatcgc catctgctgc acgcggaaga aggcacatggctgaatatcg 4680 acggtttcca tatggggatt ggtggcgacg actcctggag cccgtcagtatcggcggaat 4740 tccagctgag cgccggtcgc taccattacc agttggtctg gtgtcaaaaataataataac 4800 cgggcagggg ggatccgcag atccggctgt ggaatgtgtg tcagttagggtgtggaaagt 4860 ccccaggctc cccagcaggc agaagtatgc aaagcatgcc tgcagcccgggggatccact 4920 agtgtatgtt tagaaaaaca aggggggaac tgtggggttt ttatgaggggttttataaat 4980 gattataaga gtaaaaagaa agttgctgat gctctcataa ccttgtataacccaaaggac 5040 tagctcatgt tgctaggcaa ctaaaccgca ataaccgcat ttgtgacgcgagttccccat 5100 tggtgacgcg ttttgagatt tctgtcgccg actaaattca tgtcgcgcgatagtggtgtt 5160 tatcgccgat agagatggcg atattggaaa aattgatatt tgaaaatatggcatattgaa 5220 aatgtcgccg atgtgagttt ctgtgtaact gatatcgcca tttttccaaaagtgattttt 5280 gggcatacgc gatatctggc gatagcgctt atatcgttta cgggggatggcgatagacga 5340 ctttggtgac ttgggcgatt ctgtgtgtcg caaatatcgc agtttcgatataggtgacag 5400 acgatatgag gctatatcgc cgatagaggc gacatcaagc tggcacatggccaatgcata 5460 tcgatctata cattgaatca atattggcca ttagccatat tattcattggttatatagca 5520 taaatcaata ttggctattg gccattgcat acgttgtatc catatcgtaatatgtacatt 5580 tatattggct catgtccaac attaccgcca tgttgacatt gattattgactagttattaa 5640 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccgcgttacataa 5700 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccattgacgtcaata 5760 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtcaatgggtggag 5820 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgccaagtccgccc 5880 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagtacatgacctta 5940 cgggactttc ctacttggca gtacatctac gtattagtca tcgctattaccatggtgatg 6000 cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacggggatttccaagt 6060 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacgggactttcca 6120 aaatgtcgta acaactgcga tcgcccgccc cgttgacgca aatgggcggtaggcgtgtac 6180 ggtgggaggt ctatataagc agagctcgtt tagtgaaccg acttaagtcttcctgcaggg 6240 gctctaaggt aaatagggca ctcagattct gcggtctgag tcccttctctgctgggctga 6300 aaaggccttt gtaataaata taattctcta ctcagtccct gtctctagtttgtctgttcg 6360 agatcctaca gttggcgccc gaacagggac ctgagagggg cgcagaccctacctgttgaa 6420 cctggctgat cgtaggatcc ccggccaggt gtggaaagtc cccaggctccccagcaggca 6480 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgcccctaactccgc 6540 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggctgactaattt 6600 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccagaagtagtgag 6660 gaggcttttt tggaggccta ggcttttgca aaaagcttga ttcttctgacacaacagtct 6720 cgaacttaag gctagagcca ccatgattga acaagatgga ttgcacgcaggttctccggc 6780 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcggctgctctga 6840 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtcaagaccgacct 6900 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggctggccacgac 6960 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaagggactggctgct 7020 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctgccgagaaagt 7080 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggctacctgcccatt 7140 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaagccggtcttgt 7200 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaactgttcgccag 7260 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcgatgcctgctt 7320 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtggccggctggg 7380 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctgaagagcttgg 7440 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccgattcgcagcg 7500 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggggttcgaaatg 7560 accgaccaag cgacgcccaa cctgccatca cgatggccgc aataaaatatctttattttc 7620 attacatctg tgtgttggtt ttttgtgtga atcgatagcg ataaggatcgatccgcgtat 7680 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccccgacacccgc 7740 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgcttacagacaag 7800 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatcaccgaaacgcg 7860 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatgataataatgg 7920 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccctatttgtttat 7980 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctgataaatgcttc 8040 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcccttattccct 8100 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtgaaagtaaaag 8160 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctcaacagcggta 8220 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcacttttaaagttc 8280 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactcggtcgccgca 8340 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaagcatcttacgg 8400 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgataacactgcgg 8460 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgcttttttgcacaaca 8520 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaagccataccaa 8580 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgcaaactattaa 8640 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatggaggcggata 8700 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttattgctgataaat 8760 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggccagatggtaagc 8820 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggatgaacgaaata 8880 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtcagaccaagttt 8940 actcatatat actttagatt gatttaaaac ttcattttta atttaaaaggatctaggtga 9000 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcgttccactgag 9060 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tcctttttttctgcgcgtaa 9120 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttgccggatcaag 9180 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagataccaaatactg 9240 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagcaccgcctacat 9300 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataagtcgtgtctta 9360 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggctgaacggggg 9420 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgagatacctacagc 9480 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacaggtatccggtaa 9540 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaacgcctggtatc 9600 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttgtgatgctcgt 9660 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacggttcctggcct 9720 tttgctggcc ttttgctcac atggctcgac agatct 9756 3 1964 DNAArtificial Sequence Description of Artificial Sequence m4070A 3atggccagaa gcaccctgag caagccaccc caggacaaaa tcaatccctg gaaacctctg 60atcgtcatgg gagtcctgtt aggagtaggg atggcagaga gcccccatca ggtctttaat 120gtaacctgga gagtcaccaa cctgatgact gggcgtaccg ccaatgccac ctccctcctg 180ggaactgtac aagatgcctt cccaaaatta tattttgatc tatgtgatct ggtcggagag 240gagtgggacc cttcagacca ggaaccgtat gtcgggtatg gctgcaagta ccccgcaggg 300agacagcgga cccggacttt tgacttttac gtgtgccctg ggcataccgt aaagtcgggg 360tgtgggggac caggagaggg ctactgtggt aaatgggggt gtgaaaccac cggacaggct 420tactggaagc ccacatcatc gtgggaccta atctccctta agcgcggtaa caccccctgg 480gacacgggat gctctaaagt tgcctgtggc ccctgctacg acctctccaa agtatccaat 540tccttccaag gggctactcg agggggcaga tgcaaccctc tagtcctaga attcactgat 600gcaggaaaaa aggctaactg ggacgggccc aaatcgtggg gactgagact gtaccggaca 660ggaacagatc ctattaccat gttctccctg acccggcagg tccttaatgt gggaccccga 720gtccccatag ggcccaaccc agtattaccc gaccaaagac tcccttcctc accaatagag 780attgtaccgg ctccacagcc acctagcccc ctcaatacca gttacccccc ttccactacc 840agtacaccct caacctcccc tacaagtcca agtgtcccac agccaccccc aggaactgga 900gatagactac tagctctagt caaaggagcc tatcaggcgc ttaacctcac caatcccgac 960aagacccaag aatgttggct gtgcttagtg tcgggacctc cttattacga aggagtagcg 1020gtcgtgggca cttataccaa tcattccacc gctccggcca actgtacggc cacttcccaa 1080cataagctta ccctatctga agtgacagga cagggcctat gcatgggggc agtacctaaa 1140actcaccagg ccttatgtaa caccacccaa agcgccggct caggatccta ctaccttgca 1200gcacccgccg gaacaatgtg ggcttgcagc actggattga ctccctgctt gtccaccacg 1260gtgctcaatc taaccacaga ttattgtgta ttagttgaac tctggcccag agtaatttac 1320cactcccccg attatatgta tggtcagctt gaacagcgta ccaaatataa aagagagcca 1380gtatcattga ccctggccct tctactagga ggattaacca tgggagggat tgcagctgga 1440atagggacgg ggaccactgc cttaattaaa acccagcagt ttgagcagct tcatgccgct 1500atccagacag acctcaacga agtcgaaaag tcaattacca acctagaaaa gtcactgacc 1560tcgttgtctg aagtagtcct acagaaccgc agaggcctag atttgctatt cctaaaggag 1620ggaggtctct gcgcagccct aaaagaagaa tgttgttttt atgcagacca cacggggcta 1680gtgagagaca gcatggccaa attaagagaa aggcttaatc agagacaaaa actatttgag 1740acaggccaag gatggttcga agggctgttt aatagatccc cctggtttac caccttaatc 1800tccaccatca tgggacctct aatagtactc ttactgatct tactctttgg accttgcatt 1860ctcaatcgat tggtccaatt tgttaaagac aggatctcag tggtccaggc tctggttttg 1920actcagcaat atcccagcta aaacccatag agtacgagcc atga 1964 4 63 DNAArtificial Sequence Description of Artificial Sequence wild type MMLV 4atgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg ccccctaatc 60 ccc63 5 63 DNA Artificial Sequence Description of Artificial Sequencemutant env (m4070A) 5 atggccagaa gcaccctgag caagccaccc caggacaaaaatccctggaa acctctgatc 60 gtc 63 6 14 DNA Artificial Sequence Descriptionof Artificial Sequence primer 6 tattaataac tagt 14 7 42 DNA ArtificialSequence Description of Artificial Sequence primer 7 gctacgcagagctcgtttag tgaaccgggc actcagattc tg 42 8 36 DNA Artificial SequenceDescription of Artificial Sequence primer 8 gctgagctct agagtccttttcttttacaa agttgg 36 9 18 DNA Artificial Sequence Description ofArtificial Sequence primer 9 gcattaaagc tttgctct 18 10 24 DNA ArtificialSequence Description of Artificial Sequence primer 10 gcctcgagcaaaaattcaga cgga 24 11 33 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 11 caaccaccgg gaggcaagctggccagcaac tta 33 12 39 DNA Artificial Sequence Description ofArtificial Sequence primer 12 atcggctagc agatcttcaa tattggccat tagccatat39 13 44 DNA Artificial Sequence Description of Artificial Sequenceprimer 13 atcgagatct gcggccgctt acctgcccag tgcctcacga ccaa 44 14 54 DNAArtificial Sequence Description of Artificial Sequence primer 14atcggcggcc gcccaccatg gaactcagcg tcctcctctt ccttgcaccc tagg 54 15 52 DNAArtificial Sequence Description of Artificial Sequence primer 15atcggcggcc gcacttacct gtgtgcccca ggaaagtatt tcaagaagcc ag 52 16 92 DNAArtificial Sequence Description of Artificial Sequence primer 16actgtgatca taggcaccta ttggtcttac tgacatccac tttctctcca caggcaagtt 60tacaaaacct gcaggaaatc aatgcttaca tt 92 17 41 DNA Artificial SequenceDescription of Artificial Sequence primer 17 actgatcgat ttccctcagccccttcagcg gggcaggaag c 41 18 33 DNA Artificial Sequence Description ofArtificial Sequence primer 18 gactacgact agtgtatgtt tagaaaaaca agg 33 1932 DNA Artificial Sequence Description of Artificial Sequence primer 19ctaggctact agtactgtag gatctcgaac ag 32 20 33 DNA Artificial SequenceDescription of Artificial Sequence primer 20 gggctatatg agatcttgaataataaaatg tgt 33 21 47 DNA Artificial Sequence Description ofArtificial Sequence primer 21 ttcgatgatc accaccatgg aactcagcgtcctcctcttc cttgcac 47 22 46 DNA Artificial Sequence Description ofArtificial Sequence primer 22 ttcgagccgg ctcatcagcg gggcaggaagcggatctggt atgttg 46 23 82 DNA Artificial Sequence Description ofArtificial Sequence pLTR 23 cgttaacact agtaagcttg ctctaaggta aatagtcgacaggcctgcgc cagtcctccg 60 attgactgag tcgcccgggt ac 82 24 83 DNAArtificial Sequence Description of Artificial Sequence pLTR 24cccgggcgac tcagtcaatc ggaggactgg cgcaggcctg tcgactattt accttagagc 60aagcttacta gtgttaacgg cgc 83 25 124 DNA Artificial Sequence Descriptionof Artificial Sequence pL-SA-N 25 gatctaacct aggtctcgag tgtttaaacactgggcttgt cgagacagag aagactcttg 60 cgtttctgat aggcacctat tggtcttactgacatccact ttgcctttct ctccacaggt 120 gagg 124 26 120 DNA ArtificialSequence Description of Artificial Sequence pL-SA-N 26 cctcacctgtggagagaaag gcaaagtgga tgtcagtaag accaataggt gcctatcaga 60 aacgcaagagtcttctctgt ctcgacaagc ccagtgttta aacactcgag acctaggtta 120 27 99 DNAArtificial Sequence Description of Artificial Sequence pL-SA-N with asplice donor deletion 27 ttagctaact agtacagacg caggcgcata acatcaaacatagacactag acaatcggac 60 agacacagat aagttgctgg ccagcttgcc tcccggtgg 9928 27 DNA Artificial Sequence Description of Artificial Sequence pL-SA-Nwith a splice donor deletion 28 ccctcactcg gcgcgccagt cttccga 27 29 79DNA Artificial Sequence Description of Artificial Sequence CMV/Rjunction of EIAV LTR plasmid 29 agcagagctc gtttagtgaa ccgacttaagtcttcctgca ggggctctaa ggtaaatagg 60 gcactcagat tctgcggtc 79 30 33 DNAArtificial Sequence Description of Artificial Sequence CMV/R junction ofEIAV LTR plasmid 30 cacacctggc cggggatcct acgatcagcc agg 33 31 129 DNAArtificial Sequence Description of Artificial Sequence pEGASUS-1 31tcgacgttta aacactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac 60ctattggtct tactgacatc cactttgcct ttctctccac aggtcacgtg aagctagcct 120cgagttggc 129 32 128 DNA Artificial Sequence Description of ArtificialSequence pEGASUS-1 32 tcagccaact cgaggctagc ttcacgtgac ctgtggagagaaaggcaaag tggatgtcag 60 taagaccaat aggtgcctat cagaaacgca agagtcttctctgtctcgac aagcccagtg 120 tttaaacg 128 33 32 DNA Artificial SequenceDescription of Artificial Sequence EIAV vector 33 aggaggacag gcaagatgggagaccctttg ac 32 34 24 DNA Artificial Sequence Description of ArtificialSequence EIAV vector 34 ggggtcgact ctagagtcct tttc 24 35 32 DNAArtificial Sequence Description of Artificial Sequence EIAV vector 35gtcaaagggt ctcccatctt gcctgtcctc ct 32 36 25 DNA Artificial SequenceDescription of Artificial Sequence EIAV vector 36 ctatataagc agagctcgtttagtg 25

What is claimed is:
 1. A retroviral vector comprising: (a) a 3′ and 5′long terminal repeat (LTR); (b) a functional splice donor site withinthe 5′ LTR; (c) a functional splice donor site; (d) a first nucleotidesequence of interest (NOI) flanked upstream by the functional splicedonor site and downstream by the functional splice acceptor site; and(e) a second NOI downstream of the functional slice acceptor site andupstream of the 3′ LTR; whereby the first NOI is capable of beingspliced out of RNA when transcribed from the retroviral vector.
 2. Theretroviral vector according to claim 1 wherein the second NOI encodes atherapeutic expression product.
 3. The retroviral vector according toclaim 1 wherein the first NOI, or an expression product thereof,comprises a selectable marker or a viral element.
 4. The retroviralvector according to claim 1 wherein the functional splice donor site isfrom a virus.
 5. The retroviral vector according to claim 1 wherein thefunctional splice donor site is from an intron.
 6. The retroviral vectoraccording to claim 5 wherein the intron is the small t-intron of SV40virus.
 7. The retroviral vector according to claim 1 further comprisinga multiple cloning site, wherein the functional splice acceptor site islocate upstream of the multiple cloning site.
 8. The retroviral vectoraccording to claim 1 wherein the functional splice acceptor site is froma nucleotide sequence coding for an immunological protein.
 9. Theretroviral vector according to claim 8 wherein the immunological proteinis an immunoglobulin.
 10. Tho retroviral vector according to claim 9wherein the immunoglobulin is from an immunoglobulin heavy chainvariable region.
 11. The retroviral vector according to claim 1 whereinthe vector is a murine oncoretrovirus vector or a lentivirus vector. 12.The retroviral vector according to claim 11 wherein the vector is aMMLV, MSV, MMTV, HlV-1 or EIAV retroviral vector.
 13. A method ofproducing a retroviral vector comprising a functional splice donor sitewithin its 5′ long terminal repeat (LTR), the method comprising: (a)introducing, into a packaging cell, a retroviral pro-vector comprising:(i) a 3′ and 5′ L′ LTR; (ii) a functional splice donor site locatedwithin the 3′ LTR; (iii) a functional splice acceptor site upstream ofthe splice donor site; (iv) a first nucleotide sequence of interest(NOI) upstream of the functional splice acceptor site, wherein the firstNOI comprises a packaging signal; and (v) a second NOI downstream of thefunctional splice acceptor site and upstream of the 3′ LTR, wherein theretroviral pro-vector is packaged into a viral particle in the packagingcell; and (b) infecting a target cell with the viral particle, whereinthe retroviral pro-vector is reverse transcribe; thereby producing aretroviral vector comprising a functional splice donor site within its5′ LTR.
 14. The method according to claim 13 wherein the first NOI isexpressed in the packaging cell.
 15. The method according to claim 13wherein the first NOI further comprises a selectable marker or a viralelement.
 16. The method according to claim 15 wherein the viral elementis a retroviral envelope sequence.
 17. The method according to claim 13wherein the retroviral pro-vector is a murine oncoretrovirus pro-vectoror a lentivirus retroviral pro-vector.
 18. The method according to claim17 wherein the retroviral pro-vector is a MMLV, MSV, MMTV, HIV-1, orEIAV retroviral pro-vector.
 19. The method according to claim 13 whereinthe retroviral pro-vector comprises a non-retroviral transcriptionalcontrol sequence upstream of the functional splice donor site.
 20. Themethod according to claim 19, wherein the transcriptional controlsequence is an internal promoter.
 21. The method according to claim 19,wherein the transcriptional control sequence is located in the 5′ LTR.22. The method according to claim 19, wherein the transcriptionalcontrol sequence is located in the 3′ LTR.
 23. A retroviral vectorcomprising a functional splice donor site with its 5′ LTR, wherein theretroviral vector is produced by the method of claim
 13. 24. Aretroviral vector comprising: (a) a 3′ and 5′ long terminal repeat(LTR); (b) a functional splice donor site located within the 5′ LTR; (c)a functional splice acceptor site located downstream of the functionalsplice donor site; and (d) an NOI downstream of the functional spliceacceptor site and upstream of the 3′ LTR; whereby an interveningsequence between the functional splice donor site and the functionalsplice acceptor site spliced out of RNA transcribe from the retroviralvector.
 25. The retroviral vector according to claim 24, wherein theintervening sequence comprises a viral element.
 26. The retroviralvector according to claim 25, wherein the viral element is a packagesignal.
 27. The retroviral vector according to claim 24, wherein thefunctional splice donor site is from a virus.
 28. The retroviral vectoraccording to claim 24, wherein the functional splice donor site is froman intron.
 29. The retroviral vector according to claim 28, wherein theintron is the small t-intron of SV40 virus.
 30. The retroviral vectoraccording to claim 24, wherein the comprising a multiple cloning site,wherein the functional splice acceptor site is located upstream of themultiple cloning site.
 31. The retroviral vector according to claim 24,wherein the functional splice acceptor site is from a nucleotidesequence coding for an immunological protein.
 32. The retroviral vectoraccording to claim 31, wherein the immunological protein is animmunoglobulin.
 33. The retroviral vector according to claim 32, whereinthe immunoglobulin is from an immunoglobulin heavy chain variableregion.
 34. The retroviral vector according to claim 24, wherein thevector is a murine oncoretrovirus vector or a lentivirus vector.
 35. Theretroviral vector according to claim 34, wherein the vector is a MMLV,MSV, MMTV, HIV-1 or EIAV retroviral vector.
 36. A method of producing aretroviral vector comprising a functional splice donor site within its5′ LTR, the method comprising: (a) introducing into a packaging cell, aretroviral pro-vector comprising: (i) a 3′ and 5′ LTR; (ii) a functionalsplice donor site located within the 3′ LTR; (iii) a functional spliceacceptor site; (iv) a packaging signal upstream of the functional spliceacceptor site; and (v) an NOI downstream of the functional spliceacceptor site and upstream of the 3′ LTR, wherein the retroviralpro-vector is packaged into a viral particle in the packaging cell; and(b) infecting a target cell with the viral particle, wherein theretroviral pro-vector is reverse transcribed; thereby producing aretroviral vector comprising a functional splice donor site within its5′ LTR.
 37. The method according to claim 36, wherein the retroviralpro-vector comprises a non-retroviral transcriptional control sequenceupstream of the functional splice donor site.
 38. The method accordingto claim 37, wherein the transcriptional control sequence is an internalpromoter.
 39. The method according to claim 37, wherein thetranscriptional control sequence is located in the 5′ LTR.
 40. Themethod according to claim 37, wherein the transcriptional controlsequence is located in the 3′ LTR.
 41. A retroviral vector comprising afunctional splice donor site within its 5′ LTR, wherein the retroviralvector is produced by the method of claim 36.